Clinical Urine Specimens Research Articles

Investigating trends in antibiotic resistance of Escherichia coli isolated from clinical urine specimens in the Orkney Islands.

Urinary tract infections (UTIs) are extremely common, affecting people of all ages and health statuses. Although UTIs do not usually cause severe illness, in some cases they can lead to more serious complications, especially if their initial treatment is ineffective due to antimicrobial resistance (AMR). AMR is an increasing issue, exacerbated by misdiagnosis and inappropriate prescribing of antibiotics, thus facilitating further resistance. The aim of this study was to investigate the rates of AMR in Escherichia coli isolated from clinical urine specimens tested at the Balfour Hospital, Orkney, and determine trends related to patient risk factors. Antibiotic susceptibilities were tested for 100 isolates of uropathogenic E. coli using the VITEK 2 Compact (bioMérieux), and data were analysed using percentage resistance rates. Resistance rates were compared by patient sex, age and source (hospital versus community). The findings showed higher AMR in males compared with females, particularly for trimethoprim (TMP), with 52% in males and only 12% in females. AMR tended to be higher in E. coli isolated from hospital inpatients than from community specimens, except for amoxicillin (AMX) and co-amoxiclav. Finally, the study found that AMR of E. coli isolates was greater in patients aged over 50 than 18-50 years old, particularly for AMX and TMP. The highest resistance rates across all patient demographics were for AMX, implying that the use of this antibiotic for the treatment of E. coli UTIs is not appropriate.

Multiwavelength Surface-Enhanced Raman Scattering Fingerprints of Human Urine for Cancer Diagnosis.

Label-free surface-enhanced Raman spectroscopy (SERS) is capable of capturing rich compositional information from complex biosamples by providing vibrational spectra that are crucial for biosample identification. However, increasing complexity and subtle variations in biological media can diminish the discrimination accuracy of traditional SERS excited by a single laser wavelength. Herein, we introduce a multiwavelength SERS approach combined with machine learning (ML)-based classification to improve the discrimination accuracy of human urine specimens for bladder cancer (BCa) diagnosis. This strategy leverages the excitation-wavelength-dependent SERS spectral profiles of complex matrices, which are mainly attributed to wavelength-related vibrational changes in individual analytes and differences in the variation ratios of SERS intensity across different wavelengths among various analytes. By capturing SERS fingerprints under multiple excitation wavelengths, we can acquire more comprehensive and unique chemical information on complex samples. Further experimental examinations with clinical urine specimens, supported by ML algorithms, demonstrate the effectiveness of this multiwavelength strategy and improve the diagnostic accuracy of BCa and staging of its invasion with SERS spectra from increasing numbers of wavelengths. The multiwavelength SERS holds promise as a convenient, cost-effective, and broadly applicable technique for the precise identification of complex matrices and diagnosis of diseases based on body fluids.

Distribution characteristics of integrons and correlation analysis of antibiotic resistance in urine isolated Enterobacter cloacae.

This study aims to understand the distribution of integrons among Enterobacter cloacae isolated from clinical urine specimens in our hospital, as well as the molecular characteristics of the variable region resistance gene cassette of integron-positive strains and its relationship with drug resistance. We collected a total of 80 strains of Enterobacter cloacae isolated from urine specimens of hospitalized patients in our hospital between August 2019 and July 2023, and conducted drug sensitivity testing on them. Polymerase Chain Reaction (PCR) technology was employed to screen these strains for Class 1, 2, and 3 integrons. Following this, the promoter and variable regions of integron-positive strains were amplified and sequenced. Additionally, Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) was utilized for homology analysis of integron-positive strains. Among the 80 clinical strains, Class 1 integrons were detected in 31 (38.8%) strains, and the following resistance gene cassettes were identified: aadA2, aadA1, aadB, aac(6'), and catB8. Three types of variable region promoters were observed: PcS (4 strains), PcW (7 strains), and PcH1 (17 strains), with consistently inactive downstream P2 promoters. Additionally, Class 2 integrons were detected in 5 (6.3%) strains, carrying the variable region resistance gene cassette dfrA1-sat2-aadA1. The promoters for Class 2 integrons were uniformly of the Pc2D-Pc2A-Pc2B-Pc2C type. No Class 3 integrons were detected. The strains containing integrons showed significantly higher resistance rates to ciprofloxacin, compound sulfamethoxazole, levofloxacin, gentamicin, amikacin, and tobramycin compared to those without integrons (P<0.05). 35 strains of Enterobacter cloacae carrying integrons are primarily classified into three genotypes: A, B, and C. These genotypes are mainly distributed in the urology department and Intensive Care Unit (ICU). The distribution of variable region gene boxes and promoter types is relatively concentrated in the same genotype. Our study confirmed that Enterobacter cloacae isolated from urine samples predominantly carries Class 1 integrons with an extended array of antibiotic-resistant genes. For future research, it is recommended to explore additional resistance mechanisms and evaluate the effectiveness of new therapeutic strategies. Clinicians should be vigilant about the possibility of clonal dissemination and implement enhanced infection control measures in hospital settings.

Evaluation of a Benzodiazepine Immunoassay for Urine Drug Testing in Clinical Specimens.

Benzodiazepines are commonly prescribed medications frequently linked to instances of abuse and overdose. Historically, FDA-cleared benzodiazepine urine immunoassays cross-react poorly with glucuronidated benzodiazepine metabolites, leading to false negatives. Clinical laboratories have addressed this deficiency by creating laboratory-developed tests (LDTs) that incorporate a beta-glucuronidase hydrolysis step to increase the clinical sensitivity of these assays. Performance characteristics of 2 FDA-cleared benzodiazepine urine immunoassays (Benzodiazepines Plus, no glucuronidase and Benzodiazepines II, with glucuronidase; Roche Diagnostics) and a previously described benzodiazepine immunoassay LDT (with glucuronidase) were evaluated using 258 clinical urine specimens. The positive immunoassay cutoff was set at 200 ng/mL of nordiazepam and results were compared to an LC-MS/MS benzodiazepine LDT. Clinical sensitivity, specificity, precision, and immunoassay cross-reactivity were determined for all 3 immunoassays. The Benzodiazepines II and LDT immunoassays exhibited greater clinical sensitivity (100% and 95.2%) compared to the Benzodiazepines Plus assay (66.7%). Clinical specificity of 100% was observed for all 3 assays. Immunoassay response of the Benzodiazepines II assay was greater across the range of concentrations tested (100-1000 ng/mL) relative to the other immunoassays and was the most sensitive immunoassay for the detection of lorazepam glucuronide. The Benzodiazepines II immunoassay demonstrated the greatest clinical and analytical sensitivity compared to the Benzodiazepines Plus and LDT immunoassays. The incorporation of beta-glucuronidase was crucial, as the Benzodiazepines II and LDT immunoassays demonstrated superior clinical sensitivity when compared to the Benzodiazepines Plus immunoassay that does not incorporate a beta-glucuronidase hydrolysis step.

Detection and diagnosis of bacterial pathogens in urine using laser-induced breakdown spectroscopy

The presence of bacterial cells from three species has been detected in clinical specimens of human urine using laser-induced breakdown spectroscopy (LIBS) by using a partial least squares discriminant analysis (PLS-DA) of 360 spectra obtained from 12 specimens of infected urine and 239 spectra obtained from eight specimens of sterile urine. Nominally sterile urine specimens obtained from four patients at a local hospital after being screened negative for the presence of bacterial pathogens were spiked with known aliquots of Escherichia coli, Staphylococcus aureus, and Enterobacter cloacae to simulate clinical urinary tract infections. Fifteen emission line intensities measured from the LIBS spectra and 92 ratios of those line intensities were used as 107 independent variables in the PLS-DA for discrimination between bacteria-containing specimens and sterile specimens. The PLS-DA models possessed a 98.3% sensitivity and a 97.9% specificity for the detection of pathogenic cells in urine when single-shot LIBS spectra were tested. To increase the signal to noise ratio, thirty spectra acquired from a single specimen were also averaged together and the averaged spectra were used to construct a model. When each averaged spectrum was withheld from the model individually for testing, the diagnostic test possessed a 100% sensitivity and a 100% specificity for the detection of bacterial cells in urine, although the number of test spectra was necessarily reduced.The entire LIBS spectrum from 200 nm – 590 nm was input into an artificial neural network analysis with principal component analysis pre-processing (PCA-ANN) to diagnose the bacterial species once detected. This PCA-ANN test possessed an overall sensitivity of 97.2%, an overall specificity of 98.6%, and an overall classification accuracy of 97.9% when using 80% of the data to build a model and withholding 20% for cross-validation testing. The PCA-ANN was also performed on each of the 12 bacteria-containing filters individually, using the other 11 filters to build the model. The average sensitivity of this test, calculated by averaging the sensitivities measured for each of the three bacterial species, was 70.9% and the average specificity was 85.5%. Based on these results, the average classification accuracy for the test when used to discriminate between the three microorganisms was 80.6%. These results indicate the potential usefulness of LIBS for rapidly detecting and possibly diagnosing urinary tract infections in a clinical setting.

Biofilm Production and Porin Permeability Activity in Clinical Isolates of Ciprofloxacin-Resistant Klebsiella pneumoniae in the Tertiary Hospital in Purwokerto, Indonesia

World Health Organization reports that the current use of ciprofloxacin as a broad-spectrum antibiotic shows a pattern of high bacterial resistance in many countries. Klebsiella pneumoniae is known as a Gram-negative bacterium that most often causes infection and is resistant to antibiotics. This study aimed to find patterns of resistance mechanisms based on biofilm production and porin permeability activity in K. pneumoniae clinical specimens from patients at RSUD Prof. Dr Margono Soekardjo Purwokerto, Indonesia. Several isolates of ciprofloxacin-resistant K. pneumoniae were isolated from clinical specimens of blood, sputum, urine, pus, stool and pleural fluid from August to October 2022. Identification and sensitivity testing of K. pneumoniae to ciprofloxacin were performed using Vitek® 2 Compact. A test for biofilm production is carried out by measuring optical density (OD) with a microplate at a wavelength of 630 nm. The porin permeability activity was determined using the value of the minimum inhibitory concentration (MIC) by the LC-MS spectrophotometry method at a wavelength of 630 nm. The results showed that 72 (47 %) isolates were resistant to ciprofloxacin. Among them, 41.3 % (24/58) of ciprofloxacin-resistant K. pneumoniae were able to produce strong biofilm. Porin permeability is indicated by MIC values of 1, 2 and 4 mg/L with bacterial cell counts of 32×106, 19×106 and 34×106 CFU/mL, respectively. This number decreased from the initial control with a bacterial cell count of 10.15×107 CFU/mL. Analysis of the correlation between the significance of biofilm production and the number of bacterial cells indicated that biofilm formation was related to the number of bacterial cells (R = 0.628, p ≤ 0.05). In conclusion, the resistance mechanism of ciprofloxacin-resistant K. pneumoniae involves biofilm production and decreased porin permeability activity. HIGHLIGHTS Major resistance mechanisms in pneumoniae decreased cell permeability from porin activity and biofilm formation Bacterial outer membrane permeability to antimicrobial substances influences inherent resistance. Bacteria produce biofilm to increase their resistance to antibiotics. Porins are non-specific diffusion transport proteins that play a part in the antibiotic resistance process The evolutionary development in the case of antibiotic resistance has an impact on the emergence of understanding in new perspective patterns on how to overcome health problems related to antibiotic resistance GRAPHICAL ABSTRACT

Association of CLDN molecules with sleep apnea hypopnea syndrome: new biomarker candidates.

Obstructive sleep apnea (OSA) is a common sleep-related breathing disorder, and has become a serious threat to public health. Intermittent hypoxia caused by OSA results in a low-grade inflammatory response that leads to impaired mucosal barrier function. Claudin (CLDN) molecules are important for the permeability of the mucosal epithelium. This study aimed to explore whether CLDN molecules can be a potential biomarker of OSA. A total of 37 healthy controls and 40 OSA patients underwent a physical assessment for OSA and filled out the STOP-Bang Questionnaire (SBQ) and Epworth Sleepiness Scale (ESS). Clinical specimens of plasma and urine were obtained to observe the difference between OSA patients and healthy controls and diagnostic accuracy of CLDN molecules for OSA. CLDN1, CLDN2, and CLDN3 molecules in plasma and urine decreased in OSA patients (both p < 0.05). The areas under the receiver operating characteristic curve (AUCs) of urinary CLDN1, plasma CLDN1, urinary CLDN2, plasma CLDN2, urinary CLDN3, and plasma CLDN3 were 0.887, 0.724, 0.779, 0.676, 0.828, and 0.665, respectively. The AUC of urinary CLDN1 + CLDN2 + CLDN3 was 0.906 (95% confidence interval (CI), 0.831-0.981). The AUC of plasma CLDN1 + CLDN2 + CLDN3 was 0.776 (95% CI, 0.645-0.878). The AUC of urinary CLDN3 + SBQ was 0.899 (95% CI, 0.832-0.967). The AUC of urinary CLDN3 + ESS was 0.896 (95% CI, 0.826-0.966). In addition, Urinary CLDN-3 was negative associated with the severity of OSA. CLDN molecules are promising as useful biomarkers for OSA, which may be related to the impaired barrier function related to OSA.

Iodide-Enhanced Perovskite Nanozyme-Based Colorimetric Platform for Detection of Urinary Nuclear Matrix Protein 22.

In the last decade, perovskite nanocrystals (PNCs) have brought extensive thinking owing to their excellent optical properties. Recently, we have uncovered the peroxidase-like activity of PNCs and used this for detecting many small molecules; however, the low enzymatic activity makes them unsuitable for fluorescence analysis, which is easily disturbed by the autofluorescence of biological media. This greatly limits their application in bioanalysis. Thus, the development of a method to facilely modulate the activity of PNCs for the instrument-free colorimetric detection is highly desirable. Herein, we demonstrated an iodide-enhanced perovskite nanozyme-based colorimetric platform for the visual assay of urinary nuclear matrix protein 22 (NMP22), a typical biomarker for the diagnosis of bladder cancer. We discovered that halogen could regulate the activity of perovskite nanozymes through a simple anion replacement reaction. Experimental analysis suggested that CsPbI3 nanocrystals (NCs) displayed 24-fold higher catalytic efficiency than classical CsPbBr3 NCs. As a proof-of-concept assay, the CsPbI3 NCs could be explored into an immunoassay for the detection of NMP22 in clinical urine specimens, resulting in a low detection limit of 0.03 U/mL. This iodide-enhanced immunoassay deepens our understanding of perovskite nanozymes and also provides great potential for bioanalysis.

In Vitro Activity of Omadacycline and Comparator Antibiotics against Extended-Spectrum Beta-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Urinary Isolates.

Limited oral antibiotic options exist for urinary tract infections (UTI) caused by ESBL-producing Enterobacterales. The aim of the study was to evaluate in vitro activity of omadacycline and comparator antibiotics against clinical ESBL-producing and non-ESBL-producing E. coli and K. pneumoniae urinary isolates. 102 isolates each of E. coli and K. pneumoniae were collected from clinical urine specimens in 2019. By design, an equal number of each species were included that tested positive and negative for ESBL production. Omadacycline MICs were determined using gradient test strips and compared to MICs of comparator antibiotics as determined by an automated broth microdilution system. Isolates were considered susceptible to omadacycline if the MIC was ≤4 µg/mL for each species. 54.9% of all ESBL-producing isolates were susceptible to omadacycline, but better susceptibility was observed for ESBL-producing E. coli (74.5%). Omadacycline MICs were 2-4 fold lower for E. coli and K. pneumoniae strains not producing ESBL. The omadacycline MIC 50 and 90 values were 4 and 16 µg/mL, respectively, for all isolates studied. 74.5% of all isolates were considered susceptible to omadacycline. MICs were generally lower for E. coli strains with MIC 50 and 90 values of 4 and 8 µg/mL, respectively (87.3% susceptible), compared with K. pneumoniae. Overall, the most active agents were omadacycline and nitrofurantoin, while other comparator antibiotics were less active. Omadacycline represents a promising oral antibiotic for treating UTI caused by ESBL-producing E. coli, particularly when resistance limits other oral options. Prospective, controlled clinical trials are needed to validate these in vitro results.

Sensitive Urine Immunoassay for Visualization of Lipoarabinomannan for Noninvasive Tuberculosis Diagnosis.

Lipoarabinomannan (LAM) is a prospective noninvasive biomarker for tuberculosis (TB) diagnosis. Here, we report a visual immunoassay of high sensitivity for detecting LAM in urine samples toward TB diagnosis. This method uses a DNA-linked immunosorbent of LAM, followed by a transduction cascade into amplified visual signals using quantum dots (QDs) and calcein reaction with Cu2+ and copper nanoparticles (Cu NPs). The limit of detection (LOD) for LAM in the urine reaches 2.5 fg/mL and 25 fg/mL using a fluorometer and length readouts on strips, respectively, demonstrating an ultrahigh sensitivity. The clinical validation of the proposed assay was performed with 147 HIV-negative clinical urine specimens. The results show the sensitivity of test is 94.1% (16/17) for confirmed TB (culture-positive) and 85% (51/60) for unconfirmed TB (clinical diagnosis without positive culture results), respectively, when the test cutoff value is 40 fg/mL for TB. Its specificity is 89.2% (25/28) in non-TB and nontuberculous mycobacterial patients. The area under the curve (AUC) was 0.86 when controls were non-TB and LTBI patients, while the AUC was 0.92 when controls were only non-TB patients. This highly sensitive visual immunoassay of LAM has shown potential for noninvasive diagnosis of TB using urine samples.

The association between biofilm formation ability and antibiotic resistance phenotype in clinical isolates of gram-negative bacteria: a cross-sectional study

Backgrounds: Antimicrobial-resistant pathogens that form biofilms cause treatment failure, increased mortality, and morbidity. Gram-negative bacteria (GNB) infections have become an emerging issue in antimicrobial resistance. This study analyzes the association between GNB biofilm formation ability and the antibiotic resistance phenotype in GNB clinical isolates. Methods: From June to August 2022, this cross-sectional study was conducted at Dr. Soetomo General Academic Hospital. Isolates derived from clinical specimens, including blood, urine, and respiratory tract specimens, were identified for antibiotic resistance phenotypes along with a quantitative biofilm formation assay. The antibiotic resistance phenotype was studied in relation to the GNB isolates' ability for biofilm formation. Results: Of the 271 isolates studied, 95 (35.1%) were non-MDR, 143 (52.8%) were MDR, and 33 (12.2%) were XDR. On the biofilm formation assay, 34 (12.5%) were non-biofilm producers, 237 (87.5%) were biofilm producers, consisting of 101 (37.3%) weak, 131 (48.3%) moderate, and 5 (1.8%) strong biofilm producers. Biofilm formation and antibiotic resistance in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii were not significantly associated (p &gt; 0.05). However, Escherichia coli isolates resistant to the antibiotics amikacin, imipenem, meropenem, and amoxicillin-clavulanate were found to have a higher predisposition to form biofilms. Klebsiella pneumoniae isolates resistant to gentamycin were also positively associated with biofilm formation. Conclusions: Gram-negative bacteria's ability to form biofilms is not associated with their antibiotic resistance phenotype; therefore, routine biofilm monitoring is critical for enhancing the quality of treatment strategy for biofilm-associated infection.

Comparison of two highly sensitive benzodiazepine immunoassay lab developed tests for urine drug testing in clinical specimens

BackgroundThe VALID Act is a legislative effort that, if enacted, would alter the regulatory requirements of laboratory developed tests (LDTs) used for clinical testing in the United States. Benzodiazepines, which are primarily excreted into urine as glucuronidated metabolites such as lorazepam, cross-react poorly with FDA-cleared immunoassays, leading to false-negatives. This shortfall can be addressed with LDTs created by adding glucuronidase to the immunoassay reagents producing “high sensitivity” assays that detect glucuronidated metabolites. MethodsPrecision and stability of two high-sensitivity (HS) benzodiazepine immunoassays from Roche and Thermo Scientific were evaluated using manufacturer-supplied quality control (QC) material and glucuronidated QC material. The immunoassays were directly compared to an LC-MS/MS LDT benzodiazepine assay to determine clinical sensitivity/specificity using urine specimens (n = 82 for Thermo Scientific; n = 265 for Roche). The clinical impact of the HS LDT immunoassay was determined by analyzing clinical testing results 60 days before and after its implementation. ResultsThe precision and clinical sensitivity/specificity of the HS-Thermo Scientific and HS-Roche benzodiazepine assays were acceptable. The reagent stability of the HS-Thermo Scientific immunoassay was poor, whereas the HS-Roche immunoassay was stable. After implementation of the HS-Roche benzodiazepine immunoassay as an LDT, there was a 30-fold increase (p-value: < 0.00001) in the percentage of lorazepam confirmations. ConclusionsWe demonstrate the development and validation of an immunoassay LDT with improved sensitivity for glucuronidated benzodiazepines. This LDT can detect glucuronidated benzodiazepines in clinical urine specimens and is stable for 60 days. Importantly, we were able to validate the immunoassay as an LDT by utilizing an LC-MS/MS LDT.

322. Clinical-Grade Metagenomics in Urinary Tract Infections: Improving Performance of Next-Generation Sequencing Assays Using Internal Controls and Machine Learning

Abstract Background Shotgun sequencing-based metagenomics is a useful approach to profiling microbiomes in environmental and patient samples. In a clinical setting, metagenomic techniques have the advantage of identifying organisms, which cannot be readily cultured or confirmed by other techniques. We have developed a clinical-grade, streamlined metagenomics-based pipeline that includes regulatory compliant method considerations, such as an internal control followed by a machine-based learning (ML) process to identify pathogens in urine samples. Methods We built an optimized novel end-to-end NGS assay pipeline that harnesses pathogen-specific genome data to detect bacterial species. We processed de-identified clinical urine specimens, collected from patients symptomatic for urinary tract infection (UTI). This workflow includes an IPC, QIACube-MDx extraction, library preparation and Illumina NextSeq 550 sequencing and a novel interpretable ML based analytic approach, Biotia-DX. Clinical culture results and qPCR were used as a baseline for the assay to train the ML model and to establish accuracy relative to the clinical standard of care. Results We clinically validated over 40 key uropathogens and conducted clinical studies of specificity, intra/inter reproducibility, accuracy in urine specimens (n=300), and limit of detection in E. coli, K. pneumoniae, P. mirabilis, S. aureus, E. faecalis and Candida. Additionally, the implementation of an internal control coupled with our Biotia-DX software provides an accurate (F1 score 94.3%) and highly sensitive clinical grade diagnostic tool. Conclusion Urine has historically presented a challenge for diagnostics via culturing, with a high rate of culture-negative results (∼30% on average). We improved the clinical utility of an NGS urine assay by leveraging an IPC and ML software. This decreased the rate of false positive species called in a sample relative to other NGS techniques and allows for greater sensitivity and taxonomic specificity. This assay may be especially useful for low colony-count or negative-culture samples to diagnose and guide patient treatment. Disclosures Mara Couto-Rodriguez, MS, Biotia Inc.: Employee of Biotia Inc. a for-profit biotechnology company David C. Danko, PhD, Biotia Inc.: Employee of Biotia Inc. a for-profit biotechnology company Xavier O. Jirau Serrano, MS, Biotia Inc.: Employee of Biotia Inc. a for-profit biotechnology company Taylor Paisie, MS, Biotia Inc.: Employee of Biotia Inc. a for-profit biotechnology company John C. Papciak, BS, Biotia Inc.: Employee of Biotia Inc. a for-profit biotechnology company Eszter Szollosi, BS, Biotia Inc.: Employee of Biotia Inc. a for-profit biotechnology company Christopher E. Mason, PhD, Biotia Inc.: Advisor/Consultant|Biotia Inc.: Board Member|Biotia Inc.: Ownership Interest Caitlin Otto, PhD, D(ABMM), Biotia Inc.: Advisor/Consultant Niamh B. O'Hara, PhD, Biotia Inc.: Board Member|Biotia Inc.: Ownership Interest Dorottya Nagy-Szakal, MD PhD, Biotia Inc.: Employee of Biotia Inc. a for-profit biotechnology company|Biotia Inc.: Stocks/Bonds.

Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis.

Lipoarabinomannan (LAM), a component of the Mycobacterium tuberculosis (MTB) cell wall, is detectable in the urine of MTB infected patients with active tuberculosis (TB). LAM-specific antibodies (Igs) have been developed by a variety of traditional and recombinant methods for potential use in a rapid diagnostic test (RDT). We evaluated the analytical performance of the TB LAM Igs to identify pairs that offer superior performance over existing urine LAM tests. We assessed 25 new and 4 existing Igs in a matrixed format using a multiplex electrochemiluminescence-based liquid immunoassay. A total of 841 paired Ig combinations were challenged with in vitro cultured LAM (cLAM) derived from MTB strains representing diverse phylogenetic lineages, alongside urinary LAM (uLAM) from the urine of adults with active pulmonary TB. Analytical sensitivity of down-selected Ig pairs was determined using MTB Aoyama-B cLAM, while diagnostic accuracy was determined using clinical samples. When testing cLAM, the reactivity of Ig pairs was similar across MTB lineages 1–4 but lineage 5:6 had significantly more reactivity among Ig pairs. Overall, 41 Ig pairs had a strong binding affinity to cLAM, as compared to the reference pair of S4-20/A194-01, and 28 Ig pairs therein exhibited a strong affinity for both cLAM and uLAM. Retrospective testing on clinical urine specimens demonstrated varying sensitivities (12–80%) and specificities (14–100%). The five top pairs had a similar analytical limit of detection to the reference pair but in four instances, the sensitivity and specificity with clinical uLAM samples was poor. Overall, epitopes presented by uLAM are different from cLAM, which may affect antibody performance when testing uLAM in patient samples. Several new Ig pairs had similar ranges of high sensitivity to cLAM but overall, there were no new candidate Ig pairs identified in this round of screening with increased performance with uLAM as compared to an existing optimal pair.

Role of some proteins in resistance of clinical Acinetobacter baumannii isolates to imipenem

Acinetobacter baumannii is one of the ESKAPE pathogens which are the leading cause of nosocomial infections throughout the world. The aim of this study is to detect the role of Some Proteins in Resistance of Acinetobacter baumannii to imipenem. The research included the collection of 100 different clinical specimens of( urine, burns, and wounds) isolated from patients in Al-Diwaniyah Teaching Hospital for the period from September to December 2021. 20 isolates out of 100 isolates belonging to A.baumannii were obtained. The samples were collected from different clinical specimens distributed as follows:7(35%) Swabs of burns,8(40%) swabs of wounds, and 5(25%)from urine, an examination was conducted for (8)Antibiotics by ( Antibiotic Susceptibility Test-AST ) on 20 isolates. The results showed that all isolates are resistant to antibiotics except for imipenem showed a sensitivity of 20% and resistance of 80% to imipenem. The results of the Minimum Inhibitory Concentration level of imipenem that were conducted for five isolates showed that all isolates are resistant to imipenem at concentrations of (128 mg/ml and 256 mg/ml ), while 2(40%) isolates out of 5 isolates were resistant to imipenem in the concentration of 64 mg /ml.

Molecular Detection of blaSHV-la Gene in Klebsiella pneumonia Isolated from Urinary Tract Infections, Najaf, Iraq.

Klebsiella pneumoniae is a gram-negative bacterium that causes serious illnesses, including pneumonia, liver abscess, meningitis, bloodstream infections, and urinary tract infections (UTIs). This study aimed to isolate and diagnose K. pneumoniae from clinical specimens of urine from patients with UTIs and perform molecular detection of the blaSHV-la gene in K. pneumonia in the Najaf Province, Iraq. The study included 100 clinical specimens from October 2021 to March 2022. As an initial diagnosis, K. pneumoniae isolates were diagnosed based on culture and biochemical features. Apart from the usage of polymerase chain reaction (PCR) technology to identify the blaSHV-la gene, the final diagnostic was achieved by the automated Vitek-2 compact system. The biochemical findings revealed that 40 out of every 100 isolates tested positive for K. pneumoniae. These results were validated by Vitek, which revealed that 40/100 of the samples tested positive for K. pneumoniae, and by PCR utilizing the blaSHV-la gene, which showed that 13/40 of the samples tested positive for K. pneumoniae isolated from the urine of patients with UTIs. In conclusion, the results indicated that the use of the Vitek-2 technique was required to confirm the accurate identification of the pathogen. Klebsiella pneumoniae clinical isolates showed multidrug resistance to antibiotics commonly used to treat UTIs. The blaSHV gene encoded for Extended-spectrum beta-lactam antibiotic was found almost in K. pneumoniae isolates.

Assessment of the Urinary Microbiota of MSM Using Urine Culturomics Reveals a Diverse Microbial Environment.

The urinary tract is not sterile and is populated by microbial communities that influence urinary health. Men who have sex with men (MSM) are understudied yet have increased risk factors for genitourinary infections. Our objective was to interrogate the composition of MSM urinary microbiota. Midstream urine specimens (n = 129) were collected from MSM (n = 63) and men seen for routine care (clinical cohort, n = 30). Demographics and sexual/medical history were documented. Specimens underwent culture using standard-of-care and enhanced methods designed to isolate fastidious and anaerobic microorganisms. Isolates were identified by MALDI-TOF mass spectrometry or 16S rRNA gene sequencing. The MSM cohort was younger (mean (SD), 35.4 (11.26) years) compared to the clinical cohort (62.7 (15.95) years). Organism recovery was significantly increased using enhanced vs standard culture for the MSM (mean of 9.1 vs 0.6 species/sample [P < 0.001]) and clinical (7.8 vs 0.9 species/sample [P < 0.001]) cohorts. The microbial composition of MSM urine specimens was dominated by Gram-positive and anaerobic microbes and clustered distinctly from that of clinical urine specimens. Composition of microbial species recovered within the same subject was dynamic between urine specimens but more similar relative to inter-individual comparisons. Principal coordinate analysis showed no correlation between urinary microbiota composition and age, recent antibiotic use, sexually transmitted infection/HIV status, or sexual practices. Enhanced culture recovered a large diversity of microbial species from MSM urine specimens, especially taxa typically associated with mucosal surfaces. These findings may increase understanding of urologic disease in MSM and improve diagnostic methods for detection of genitourinary infections.

Factors Compromising Glucuronidase Performance in Urine Drug Testing Potentially Resulting in False Negatives.

Next generation β-glucuronidases can effectively cleave glucuronides in urine at room temperature. However, during the discovery studies, additional challenges were identified for urine drug testing across biologically relevant pH extremes and patient urine specimens. Different enzymes were evaluated across clinical urine specimens and commercially available urine control matrices. Each enzyme shows distinct substrate preferences, pH optima, and variability across clinical specimens. These results demonstrate how reliance on a single glucuronidated substrate as the internal hydrolysis control cannot ensure performance across a broader panel of analytes. Moreover, sample specific urine properties compromise β-glucuronidases to varying levels, more pronounced for some enzymes, and thereby lower the recovery of some drug analytes in an enzyme-specific manner. A minimum of 3-fold dilution of urine with buffer yields measurable improvements in achieving target pH and reducing the impact of endogenous compounds on enzyme performance. After subjecting the enzymes to pH extremes and compromising chemicals, one particular β-glucuronidase was identified that addressed many of these challenges and greatly lower the risk of failed hydrolyses. In summary, we present strategies to evaluate glucuronidases that aid in higher accuracy urine drug tests with lower potential for false negatives.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry for Identification of Microorganisms in Clinical Urine Specimens after Two Pretreatments.

Rapid identification of microorganisms in urine is essential for patients with urinary tract infections (UTIs). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed as a method for the direct identification of urinary pathogens. Our purpose was to compare centrifugation-based MALDI-TOF MS and short-term culture combined with MALDI-TOF MS for the direct identification of pathogens in urine specimens. We collected 965 urine specimens from patients with suspected UTIs, 211/965 isolates were identified as positive by conventional urine culture. Compared with the conventional method, the results of centrifugation-based MALDI-TOF MS were consistent in 159/211 cases (75.4%), of which 135/159 (84.9%) had scores ≥ 2.00; 182/211 cases (86.3%) were detected using short-term culture combined with MALDI-TOF MS, of which 153/182 (84.1%) had scores ≥ 2.00. There were no apparent differences among the three methods (p = 0.135). MALDI-TOF MS appears to accelerate the microbial identification speed in urine and saves at least 24 to 48 hours compared with the routine urine culture. Centrifugation-based MALDI-TOF MS is characterized by faster identification speed; however, it is substantially affected by the number of bacterial colonies. In contrast, short-term culture combined with MALDI-TOF MS has a higher detection rate but a relatively slow identification speed. Combining these characteristics, the two methods may be effective and reliable alternatives to traditional urine culture.

In Vivo Generation of BK and JC Polyomavirus Defective Viral Genomes in Human Urine Samples Associated with Higher Viral Loads.

ABSTRACTDefective viral genomes (DVGs) are parasitic viral sequences containing point mutations, deletions, or duplications that might interfere with replication. DVGs are often associated with viral passage at high multiplicities of infection in culture systems but have been increasingly reported in clinical specimens. To date however, only RNA viruses have been shown to contain DVGs in clinical specimens. Here, using direct deep sequencing with multiple library preparation strategies and confirmatory digital droplet PCR (ddPCR) of urine samples taken from immunosuppressed individuals, we show that clinical BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV) strains contain widespread genomic rearrangements across multiple loci that likely interfere with viral replication. BKPyV DVGs were derived from BKPyV genotypes Ia, Ib-1, and Ic. The presence of DVGs was associated with specimens containing higher viral loads but never reached clonality, consistent with a model of parasitized replication. These DVGs persisted during clinical infection as evidenced in two separate pairs of samples containing BK virus collected from the same individual up to 302 days apart. In a separate individual, we observed the generation of DVGs after a 57.5-fold increase in viral load. In summary, by extending the presence of DVGs in clinical specimens to DNA viruses, we demonstrate the ubiquity of DVGs in clinical virology.IMPORTANCE Defective viral genomes (DVGs) can have a significant impact on the production of infectious virus particles. DVGs have only been identified in cultured viruses passaged at high multiplicities of infection and RNA viruses collected from clinical specimens; no DNA virus in the wild has been shown to contain DVGs. Here, we identified BK and JC polyomavirus DVGs in clinical urine specimens and demonstrated that these DVGs are more frequently identified in samples with higher viral loads. The strains containing DVGs had rearrangements throughout their genomes, with the majority affecting genes required for viral replication. Longitudinal analysis showed that these DVGs can persist during an infection but do not reach clonality within the chronically infected host. Our identification of polyomavirus DVGs suggests that these parasitic sequences exist across the many classes of viruses capable of causing human disease.