Female Urine Research Articles

Facile synthesis of active sites-rich magnetic adsorbent by simple one-pot hydrothermal strategy for quick and highly capable capture of female-steroid hormones

Rapid and efficient extraction is highly desired in the accurate quantification of female-steroid hormones (FSHs) in complex samples. In this context, an active sites-rich magnetic adsorbent (MAC) modified with carboxylated multiwalled carbon nanotubes and 4-vinylphenylboronic acid was facilely synthesized through one-pot hydrothermal technique for the magnetic solid phase extraction (MSPE) of FSHs in water and urine samples. Various characterizations revealed that the prepared adsorbent contained abundant functional groups, acceptable magnetic properties, high surface area and good stability. Due to the multiple interactions, the developed MAC/MSPE exhibited high isolation and preconcentration capability towards studied FSHs within 10 min. The concentration factors were 151–354 and the adsorption capacity was as high as 3.2 mg/g. Adsorption behaviors of MAC/MSPE towards FSHs were studied based on adsorption kinetics and isotherm experiments. After optimizing the extraction parameters, the MAC/MSPE was combined with HPLC to sensitive and reliable monitoring of studied FSHs in various waters and female urine samples. The limits of detection and recoveries with different spiked contents were 0.0015–0.0026 μg/L and 80.1–119 %, respectively. Accuracy of the established method was evaluated by confirmatory testing. Moreover, sample preparation greenness of the developed method was assessed. The present study uncovered the prospect of MAC/MSPE in the rapid and highly capable capture of FSHs, and also supplied a sensitive and reliable approach for the monitoring of trace FSHs in complex samples.

A-359 Clinical Performance Evaluation of the Polymerase Chain Reaction (PCR)-Based cobas CT/NG/MG Test for Use on the cobas liat System in a Clinical Laboratory Setting and Point-of-Care (POC) Location

Abstract Background Screening for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Mycoplasma genitalium (MG) can be achieved rapidly (in approximately 20 minutes) with the cobas® CT/NG/MG test (assay not cleared by US FDA. Submission currently under review and subject to change per health authority feedback). This automated, qualitative, real-time PCR-based nucleic acid amplification test (NAAT) is for use on the cobas® liat® system. This study evaluated the test’s clinical performance using urogenital samples from symptomatic and asymptomatic patients. Methods This non-interventional, non-observational study used prospective clinician-collected and self-collected specimens (urine, and vaginal swabs, all in cobas® PCR Media) from symptomatic/asymptomatic patients ≥14 years old from 13 POC sites across the US. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of cobas liat CT/NG/MG were calculated with respect to patient infected status (PIS), as determined using results from three FDA-approved NAATs and one laboratory-developed test. Due to insufficient positive NG samples during the trial period, further testing used archived samples. Results The median (range) age of the study population (N=4,800) was 35.0 (15.0-81.0) years; 40.4% (n=1,941) were symptomatic and 51.9% (n=2,489) were assigned female at birth. The cobas CT/NG/MG nucleic acid test demonstrated good clinical performance (Table 1). Across all specimen types, specificity was >97% for each analyte. Sensitivity was ≥95%, except in female urine (CT 87.0%, NG 93.1%, MG 78.9%). Regardless of specimen type, PPV and NPV were ≥95% for CT and NG; PPV for MG was highest in male urine (92.7%) and NPV was >97.5% across analytes. Conclusions In a clinical setting, the cobas CT/NG/MG nucleic acid test showed good clinical performance for the detection of CT, NG, and MG in urogenital samples, in addition to providing a short turn-around time and centralized testing lab quality at the POC for both self- and clinician-collected samples.

A-284 Development of a Low Positive Molecular Control for Mycoplasma genitalium and Trichomonas vaginalis Screening

Abstract Background Mycoplasma genitalium (MG) is a sexually transmitted gram -negative bacteria that infects the urinary and genital tracts. Trichomonas vaginalis (TV) is a protozoan parasite and is the most common curable sexually transmitted infection (STI) worldwide. TV/ MG infections often go unrecognized because infected individuals are either asymptomatic or have symptoms similar to other infections of the urogenital tract (cervicitis in females and urethritis in males). Left untreated, TV/MG can lead to severe health complications including pelvic inflammatory disease, adverse pregnancy outcomes such as preterm delivery and miscarriage, increased risk of HIV acquisition, sexually acquired reactive arthritis, and infertility. The rates of STIs are increasing and differentiating among different STIs is critical to ensure patients receive effective treatment. Amplified nucleic acid based tests (NAATs) have emerged as the gold standard for diagnosis of TV/MG infections due to their high sensitivity and high throughput using a variety of clinical samples including male and female urine. This study describes work to develop a low positive quality control material in human urine for TV/MG amplified nucleic acids tests. Methods A method was developed to eliminate residual enzymatic activity from raw, pooled human urine from both genders to create a homogeneous diluent that supports analyte stability. The urine diluent was tested for viral pathogens (HIV-1, HCV, HBV) and ensure urine proteins and chemistry analytes were within acceptable limits. TaqMan® assays for TV (targeting RNA polymerase Rpb1 gene) and MG (targeting SMC Chromosome segregation gene) were developed and qualified for digital PCR quantitation of these organisms using the BioRad QX200 system. Cultured Trichomonas vaginalis strain C-1:NIH and Mycoplasma genitalium strain G37 were spiked into the urine diluent along with 0.09% sodium azide as a preservative. The concentration of TV and MG was guided by digital PCR quantitation throughout formulation and the final product was tested on Abbott Alinity m STI Panel, Hologic Aptima® Mycoplasma genitalium Assay, and Hologic Aptima® Trichomonas vaginalis Assay. Results Three independent lots were produced that were each detected for both TV and MG on the Abbott Alinity m STI panel. The concentration of the controls targeted a cycle number (CN) of ∼27 for TV and ∼32 for MG and were within acceptable limits. The lots were also positive for TV and MG when tested on the Hologic Aptima TV and MG assays. Accelerated stability studies showed that the product is stable throughout 2 years of shelf life when stored frozen at -20 °C, but is also stable for up to 30 days at 2-8 °C refrigerated storage. Conclusions LGC Clinical Diagnostics is developing a stable, whole organism molecular control material for use with in vitro diagnostic test methods that detect TV and MG. The product will fulfill an unmet need for molecular quality controls for STI testing that are formulated in a true patient-like matrix like urine. This study helps ensure the molecular control will be a low positive on leading diagnostic assays, be consistent batch to batch, and have the needed stability to meet the needs of clinical testing labs.

A GnRH neuronal population in the olfactory bulb translates socially relevant odors into reproductive behavior in male mice.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons regulate fertility and integrate hormonal status with environmental cues to ensure reproductive success. Here we show that GnRH neurons in the olfactory bulb (GnRHOB) of adult mice can mediate social recognition. Specifically, we show that GnRHOB neurons extend neurites into the vomeronasal organ and olfactory epithelium and project to the median eminence. GnRHOB neurons in males express vomeronasal and olfactory receptors, are activated by female odors and mediate gonadotropin release in response to female urine. Male preference for female odors required the presence and activation of GnRHOB neurons, was impaired after genetic inhibition or ablation of these cells and relied on GnRH signaling in the posterodorsal medial amygdala. GnRH receptor expression in amygdala kisspeptin neurons appear to be required for GnRHOB neurons' actions on male mounting behavior. Taken together, these results establish GnRHOB neurons as regulating fertility, sex recognition and mating in male mice.

Disposable and flexible screen-printed-carbon electrode modified with Au/poly-beta-cyclodextrin as electrochemical platform for estriol detection

In this work, a screen-printed carbon electrode in polyethylene (SPPE) modified with Au particles and poly-beta-cyclodextrin polymeric film (SPPE/Au/p-beta-CD) was develop as an electrochemical platform to detect estriol (E3) in environmental and biological samples. The stability, reproducibility and sensitivity of the electrode were confirmed via physicochemical and electrochemical characterization. Electrochemical tests demonstrated an increase in the SPPE electroactive area when 70% graphite ink was applied, and transmission electron microscopy revealed an increase in surface complexity compared with that of the unmodified electrode. The electrochemical impedance spectroscopy (EIS) to SPPE/Au/p-beta-CD data indicated a decrease and increase in the Rct (0.096 kΩ cm2) and constant phase element (CPE) (28.34 µF cm−2) values, respectively. The analytical curve was plotted using concentrations ranging from 2.0 to 48.5 µmol/L and the best results were obtained at pH 4.0; a limit of detection (LD) of 1.88 µmol/L was obtained. During a study of interferents, the platform exhibited high selectivity for E3. The SPPE/Au/p-beta-CD exhibited recovery percentages of E3 between 100.32–110.17% in river water, and female urine, saliva, and sweat. The SPPE/Au/p-beta-CD as electrochemical platform proved to be a reproducible, selective, and low-cost sensor for the detection of E3 in different complex matrices. The modification of SPPE with Au and the beta-CD polymer made the electrode more sensitive and the method used is viable for real application.

Diversity and Antibiotic Susceptibility of Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae from the Urine of Human Population Using Bacteria-Contaminated Water in Butembo Urban Area (DR Congo, Central Africa)

Aims: This study aims to investigate the extended-spectrum beta-lactamases (ESBLs)-producing Enterobacteriaceae in the human urine samples and to understand the potential influence of the contaminated water the urine donors use. Study Design and Methodology: One hundred urine samples from females and the same number from males were collected from donors of different age groups and analysed. One hundred urine samples from females and the same number from males were collected from donors of different age groups. The enterobacterial isolation was done on CLED agar medium. Their identification was carried out first by the enzymatic technic, then using MALDI–TOF MS method. ESBLs-producing enterobacteria were identified by searching the synergy between Clavulanic acid and 3rd generation Cephalosporins. The antimicrobial susceptibility tests were carried out using the disk diffusion method. Results: In male urine samples, the identified ESBLs-producing strains belong to E. coli, E. agglomerans and K. pneumoniae. They were in 11 samples: 3 from donors aged 20 years or less, 3 also from those aged 21 to 40 years as well as in those aged 41 to 60 years, and 2 in samples from donors aged over than 60 years. In female urine samples, the identified ESBLs-producing strains belong to Escherichia coli, Enterobacter agglomerans, Yersinia frederiksenii, Salmonella typhi and Klebsiella pneumoniae. They were in 27 samples: 3 from donors aged 20 years or less, 9 from those aged 21 to 40 years, 12 from those aged 41 to 60 years, and 3 from donors aged over than 60 years. All the strains were sensitive to Imipenem and resistant to Cefotaxim, Ceftazidin, Amoxicillin+Clavulanic acid and Nitrofurantoin. The Multi-Antibiotic Resistance (MAR) index varied from 0.416 (E. coli strains, youngest male donors) to 0.916 (E. coli strains, old female donors). Most of strains react differently from one antibiotic to another (P<0.05), with the exception of E. coli strains from most samples. Most of the identified ESBLs-producing species have been reported in surface and groundwater the population use. These water resources could play a role in the human urinary tract infections. Conclusion: There is a serious health problem with great MAR indices for all bacterial species identified. Treating water before use could reduce the viability and spreading of ESBLs-producing bacteria and genes into the population.

Draft genome sequences of four Corynebacterium amycolatum strains isolated from female urine samples.

Corynebacterium amycolatum is an emerging pathogen of the urinary tract. Here, we present the draft genomes for four strains isolated from urine collected from symptomatic and asymptomatic female participants.

Draft genome assemblies of one Lactobacillus gasseri strain and two Lactobacillus jensenii strains isolated from voided urine samples.

We present the draft genome for three Lactobacillus strains isolated from female urine specimens: Lactobacillus gasseri UMB1673, Lactobacillus jensenii UMB1855, and Lactobacillus jensenii UMB5069. Focusing on strains within the female urinary microbiome can provide a more well-rounded understanding of the microbial community and its influence on health and disease.

Antioxidant Response of Maternal and Fetal Rat Liver to Selenium Nanoparticle Supplementation Compared to Sodium Selenite: Sex Differences between Fetuses.

To compare the effects of organic selenium nanoparticles (SeNPs, Se0) and inorganic sodium selenite (NaSe, Na2SeO3, Se4+) on the antioxidant response in maternal and fetal rat liver, pregnant females were treated with two forms of selenium (Se) at equivalent doses during gestation (0.5 mg SeNPs or 0.5 mg NaSe/kg body weight/day). Structural parameters of the liver of gravid females and their fetuses were examined in a sex-specific manner. The oxidative stress parameters superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione S-transferase (GST), total glutathione (GSH) and sulfhydryl groups (SH) were established. In addition, the Se concentration was determined in the blood, liver, urine and feces of the gravid females and in the liver of the fetuses. The structure of the liver of gravid females remained histologically the same after supplementation with both forms of Se, while the oxidative stress in the liver was significantly lower after the use of SeNPs compared to NaSe. Immaturity of fetal antioxidant defenses and sex specificity were demonstrated. This study provides a detailed insight into the differences in the bioavailability of the nano form of Se compared to sodium selenite in the livers of pregnant females and fetuses.

Basmisanil, an α5-GABAAR negative allosteric modulator, produces rapid and sustained antidepressant-like responses in male mice

There is a critical need for safer and better-tolerated alternatives to address the current limitations of antidepressant treatments for major depressive disorder. Recently, drugs targeting the GABA system via α5-containing GABAA receptors (α5-GABAAR) as negative allosteric modulators (α5-NAMs) have shown promise in alleviating stress-related behaviors in preclinical studies, suggesting that α5-NAMs may have translational relevance as novel antidepressant medications. Here, we evaluated the efficacy of Basmisanil, an α5-NAM that has been evaluated in Phase 2 clinical studies as a cognitive enhancer, in a battery of behavioral tests relevant to coping strategies, motivation, and aversion in male mice, along with plasma and brain pharmacokinetic measurements. Our findings reveal that Basmisanil induces dose-dependent rapid antidepressant-like responses in the forced swim test and sucrose splash test without promoting locomotor stimulating effects. Furthermore, Basmisanil elicits sustained behavioral responses in the female urine sniffing test and sucrose splash test, observed 24 h and 48 h post-treatment, respectively. Bioanalysis of plasma and brain samples confirms effective blood–brain barrier penetration by Basmisanil and extrapolation to previously published data suggest that effects were observed at doses (10 and 30 mg/kg i.p.) corresponding to relatively modest levels of α5-GABAAR occupancy (40–65 %). These results suggest that Basmisanil exhibits a combination of rapid and sustained antidepressant-like effects highlighting the potential of α5-NAMs as a novel therapeutic strategy for depression.

Clinical outcomes of female external urine wicking devices as alternatives to indwelling catheters: a systematic review and meta-analysis.

Female patients using indwelling urinary catheters (IUCs) are disproportionately at risk for developing catheter-associated urinary tract infections (CAUTIs) compared to males. Female external urine wicking devices (FEUWDs) have emerged as potential alternatives to IUCs for incontinence management. To assess the clinical risks and benefits of FEUWDs as alternatives to IUCs. Ovid MEDLINE, Embase, Scopus, Web of Science Core Collection, CINAHL Complete, and ClinicalTrials.gov were searched from inception to July 10, 2023. Included studies used FEUWDs as an intervention and reported measures of urinary tract infections and secondary outcomes related to incontinence management. Of 2,580 returned records, 50 were systematically reviewed. Meta-analyses assessed rates of indwelling CAUTIs and IUC utilization. Following FEUWD implementation, IUC utilization rates decreased 14% (RR = 0.86, 95% CI=[0.76, 0.97]) and indwelling CAUTI rates nonsignificantly decreased up to 32% (IRR = 0.68, 95% CI=[0.39, 1.17]). Limited only to studies that described protocols for implementation, the incidence rate of indwelling CAUTIs decreased significantly up to 54% (IRR = 0.46, 95% CI=[0.32, 0.66]). Secondary outcomes were reported less routinely. Overall, FEUWDs nonsignificantly reduced indwelling CAUTI rates, though reductions were significant among studies describing FEUWD implementation protocols. We recommend developing standard definitions for consistent reporting of non-indwelling CAUTI complications such as FEUWD-associated UTIs, skin injuries, and mobility-related complications.

Sex differences in amino acid composition in a rodent model of ARPKD, the PCK rat

Polycystic Kidney Disease (PKD) is a severe, genetically inherited disease that leads to renal failure. Like several other chronic kidney diseases, male sex is a risk factor for accelerated PKD. Rat models of both Autosomal Recessive and Dominant forms of PKD (PCK and Han:SPRD rats, respectively) exhibit sex differences in disease progression. Previous studies have found that amino acids (AAs) and peptides are predominant constituents of cystic fluid. We hypothesized that AA homeostasis in male and female cystic fluid would demonstrate sexual dimorphism, and these differences may provide essential information about the progression of cystogenesis. We quantified the AAs in cystic fluid, plasma, and urine in PCK and control Sprague Dawley (SD) rats (only plasma and urine for SD rats). Of the 42 AAs that were measured, only asparagine, serine, aspartic acid, glutamic acid, beta-alanine, cystathionine, and leucine were NOT significantly different between SD and PCK plasma values. Interestingly, only 18 AAs were significantly different in urine of PCK rats compared to SD rats. In SD rats, between sexes, 26 AAs were significantly different in plasma and 5 were significantly different in urine. Among PCK rats, 13 AAs in plasma, 9 AAs in urine, and 4 AAs in cystic fluid were significantly different between sexes. The 4 AAs that differed between male and female cystic fluid were 1-methylhistidine, 3-methylhistidine, ethanolamine, and taurine. Moreover, taurine was the most abundant AA in cystic fluid. Taurine concentrations in both plasma (165 ± 9 vs. 128 ± 4 μM, N=6, p=0.003) and cystic fluid (7508 ± 469 vs. 4557 ± 788 μM, N=6, p=0.01) were significantly different between sexes in the PCK rats (male vs. female, respectively). Urine values for the PCK rats (9705 ± 1863 vs. 7570 ± 1631 μM, N=6) were not significantly different in males and females; however, the average urinary taurine excretion in PCK rats was nearly 4-fold greater than SD rats (8637 ± 1223 vs. 2219 ± 465 μM, N=12, p<0.0001). There were no significant differences in taurine concentrations between SD male and female rat plasma (202 ± 17 vs. 194 ± 7.0 μM, N=6) or urine (2044 ± 614 vs. 2395 ± 752 μM, N=6). Western blot of the taurine transporter, TauT, detected bands at 50 and 75 kDa, and both bands were significantly reduced by ~50% in PCK kidneys compared to SD kidneys. Lastly, immunolabeling in PCK kidney sections demonstrated that TauT is present at the apical membrane of cyst epithelia. These results point to a potential role for AAs, and specifically for taurine in cystogenesis, which likely varies by sex in diseased, but not healthy kidneys. R00 HL153686, BX004024, R01 DK126720, R01 DK129227. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Seasonal investigation of the macroscopic and microscopic structure of the sinus interdigitalis in Hamdani crossbred sheep (Ovis aries).

The odours of different body parts and structures, such as external genitalia, female urine, ventral tail radix, infraorbital sinus and interdigital sinus, have a strong attractiveness for rams. The sinus interdigitalis is considered an important trail gland for sexual behaviour, as its odour is included in the list of strongly attractive structures for male animals during the breeding season, being important in pheromone production. In the present study, the effects of sex and breeding season on the morphological and histological structure of the interdigital gland were investigated in Hamdani crossbred sheep (Ovis aries). Sinus interdigitalis glands located on the forelimbs and hind limbs were collected from 10 male and 10 female sheep during both breeding and non-breeding seasons. The gland's position in the forelimbs and hind limbs was determined radiographically. The glands were examined macroscopically, histologically and immunohistochemically in both seasons. In all animals, the topographic location of the gland between the digits was determined to be distal to the phalanx proximalis and at the level of the phalanx media. The mean weight of the gland in male and female animals was 1.07±0.03 and 1.4±0.11g, respectively. In general, the results of morphometric measurements were higher during the breeding season, and the increase was more pronounced in female animals. In histological examinations, it was determined that both neck and body parts of the tissues consisted of a structure consisting of a connective tissue capsule at the outermost, dermis, epidermis and lumen underneath. Immunohistochemically staining revealed that oestrogen receptors showed positive immunoreactions only in the intracytoplasmic localization of apocrine sweat glands. In terms of morphological, histological and oestrogen receptors, it was thought that the gland may play a role in sexual communication.

Cataloging the phylogenetic diversity of human bladder bacterial isolates

BackgroundAlthough the human bladder is reported to harbor unique microbiota, our understanding of how these microbial communities interact with their human hosts is limited, mostly owing to the lack of isolates to test mechanistic hypotheses. Niche-specific bacterial collections and associated reference genome databases have been instrumental in expanding knowledge of the microbiota of other anatomical sites, such as the gut and oral cavity.ResultsTo facilitate genomic, functional, and experimental analyses of the human bladder microbiota, we present a bladder-specific bacterial isolate reference collection comprising 1134 genomes, primarily from adult females. These genomes were culled from bacterial isolates obtained by a metaculturomic method from bladder urine collected by transurethral catheterization. This bladder-specific bacterial isolate reference collection includes 196 different species, including representatives of major aerobes and facultative anaerobes, as well as some anaerobes. It captures 72.2% of the genera found when re-examining previously published 16S rRNA gene sequencing of 392 adult female bladder urine samples. Comparative genomic analysis finds that the taxonomies and functions of the bladder microbiota share more similarities with the vaginal microbiota than the gut microbiota. Whole-genome phylogenetic and functional analyses of 186 bladder Escherichia coli isolates and 387 gut Escherichia coli isolates support the hypothesis that phylogroup distribution and functions of Escherichia coli strains differ dramatically between these two very different niches.ConclusionsThis bladder-specific bacterial isolate reference collection is a unique resource that will enable bladder microbiota research and comparison to isolates from other anatomical sites.

Development and validation of a versatile non-invasive urinary steroidomics method for wildlife biomonitoring

Wildlife conservation is often challenged by a lack of knowledge about the reproduction biology and adaptability of endangered species. Although monitoring steroids and related molecules can increase this knowledge, the applicability of current techniques (e.g. immunoassays) is hampered by species-specific steroid metabolism and the requisite to avoid invasive sampling. This study presents a validated steroidomics method for the (un)targeted screening of a wide range of sex and stress steroids and related molecules in urine using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). In total, 50 steroids (conjugated and non-conjugated androgens, estrogens, progestogens and glucocorticoids) and 6 prostaglandins could be uniquely detected. A total of 45 out of 56 compounds demonstrated a detection limit below 0.01 ng μL−1. Excellent linearity (R2 > 0.99), precision (CV < 20 %), and recovery (80–120 %) were observed for 46, 41, and 39 compounds, respectively. Untargeted screening of pooled giant panda and human samples yielded 9691 and 8366 features with CV < 30 %, from which 84.1 % and 83.0 %, respectively, also demonstrated excellent linearity (R2 > 0.90). The biological validity of the method was investigated on male and female giant panda urine (n = 20), as well as pooled human samples (n = 10). A total of 24 different steroids were detected with clear qualitative and quantitative differences between human and giant panda samples. Furthermore, expected differences were revealed between female giant panda samples from different reproductive phases. In contrast to traditional biomonitoring techniques, the developed steroidomics method was able to screen a wide range of compounds and provide information on the putative identities of metabolites potentially important for reproductive monitoring in giant pandas. These results illustrate the advancements steroidomics brings to the field of wildlife biomonitoring in the pursuit to better understand the biology of endangered species.

Profiling of urinary steroids aided by lithium ion adduction-based ultrahigh-performance liquid chromatography-tandem mass spectrometry.

As 3-OH-containing steroids are prone to dehydration by conventional electrospray ionization, reducing detection sensitivity, Li ion adduction-based ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS), developed to prevent dehydration and effectively detect 3-OH steroids, was applied for profiling total and free steroids in urine. Free urinary steroids were isolated directly from urine by solid-phase extraction (SPE) with 80% acetonitrile. The total steroids were prepared by enzymatic treatment of urine with a cocktail of sulfatase and glucronidase, protein precipitation, and separation with the above SPE. In order to detect as many steroid types as possible, UHPLC/MS/MS (Li method) with Li+ solution added after the column was used for analysis in addition to the conventional method of detecting protonated ions (H method). The 13 3-OH steroids and the remaining 16 steroids were quantified by standard curves prepared using product ion transitions derived from [M + Li]+ and MH+ , respectively. Two groups of human urine, male and female urine, were analyzed. 3-OH steroids could be detected with greater sensitivity using the Li method than the conventional method. The absolute amounts of each steroid were normalized based on creatinine levels. The difference between the male and female groups are clearly attributable to sex steroids. Twenty-nine total steroids and 19 free steroids were identified in a limited volume (240 mL) of urine. Of these, 13 3-OH steroids were better detected by Li+ adduction-based UHPLC/MS/MS.

Cluster-Enhanced Nanopore Sensing of Ovarian Cancer Marker Peptides in Urine.

The development of novel methodologies that can detect biomarkers from cancer or other diseases is both a challenge and a need for clinical applications. This partly motivates efforts related to nanopore-based peptide sensing. Recent work has focused on the use of gold nanoparticles for selective detection of cysteine-containing peptides. Specifically, tiopronin-capped gold nanoparticles, trapped in the cis-side of a wild-type α-hemolysin nanopore, provide a suitable anchor for the attachment of cysteine-containing peptides. It was recently shown that the attachment of these peptides onto a nanoparticle yields unique current signatures that can be used to identify the peptide. In this article, we apply this technique to the detection of ovarian cancer marker peptides ranging in length from 8 to 23 amino acid residues. It is found that sequence variability complicates the detection of low-molecular-weight peptides (<10 amino acid residues), but higher-molecular-weight peptides yield complex, high-frequency current fluctuations. These fluctuations are characterized with chi-squared and autocorrelation analyses that yield significantly improved selectivity when compared to traditional open-pore analysis. We demonstrate that the technique is capable of detecting the only two cysteine-containing peptides from LRG-1, an emerging protein biomarker, that are uniquely present in the urine of ovarian cancer patients. We further demonstrate the detection of one of these LRG-1 peptides spiked into a sample of human female urine.

A smartphone-based immunochromatographic strip platform for on-site quantitative detection of antigenic targets.

To report the testing signal of an immunochromatographic assay for on-site quantitative detection, a portable and user-friendly smartphone-based biosensing platform is developed in this study. This innovative system is composed of an ambient light sensor inherent smartphone reader and a 3D-printed handhold device, a quantitative tool capable of directly interpreting carbon nanoparticle (CNP)-conjugated immunochromatographic strips. To showcase the platform capability, the smartphone-based immunochromatography system (SPICS) reader and device were successfully used in CNP strips for rapid detection of the early pregnancy marker human chorionic gonadotropin in female urine (HCG; limit of detection [LOD]: 0.30 mIU mL-1), prostate-specific antigen in patient blood (PSA; LOD: 0.28 ng mL-1) and ampicillin residue in animal milk (AMP; LOD: 0.23 ng mL-1). The results were fully correlated with conventional commercial instruments (R2 = 0.99). The SPICS platform exhibits significant advantages, including portability, cost-effectiveness, easy operation, and rapid and quantitative detection, making it a valuable on-site diagnosis tool for use in home and community healthcare facilities.

The Challenge to Stabilize, Extract and Analyze Urinary Cell-Free DNA (ucfDNA) during Clinical Routine.

The "Liquid Biopsy" has become a powerful tool for cancer research during the last decade. Circulating cell-free DNA (cfDNA) that originates from tumors has emerged as one of the most promising analytes. In contrast to plasma-derived cfDNA, only a few studies have investigated urinary cfDNA. One reason might be rapid degradation and hence inadequate concentrations for downstream analysis. In this study, we examined the stability of cfDNA in urine using different methods of preservation under various storage conditions. To mimic patient samples, a pool of healthy male and female urine donors was spiked with a synthetic cfDNA reference standard (fragment size 170 bp) containing the T790M mutation in the EGFR gene. Spiked samples were preserved with three different buffers and with no buffer over four different storage periods (0 h; 4 h; 12 h; 24 h) at room temperature vs. 4 °C. The preservatives used were Urinary Analyte Stabilizer (UAS, Novosanis, Wijnegem, Belgium), Urine Conditioning Buffer (UCB, Zymo, Freiburg, Germany) and a self-prepared buffer called "AlloU". CfDNA was extracted using the QIAamp MinElute ccfDNA Mini Kit (Qiagen, Hilden, Germany). CfDNA concentration was measured using the Qubit™ 4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Droplet digital PCR (ddPCR) was used for detection and quantification of the T790M mutation. Almost no spiked cfDNA was recoverable from samples with no preservation buffer and the T790M variant was not detectable in these samples. These findings indicate that cfDNA was degraded below the detection limit by urinary nucleases. Stabilizing buffers showed varying efficiency in preventing this degradation. The most effective stabilizing buffer under all storage conditions was the UAS, enabling adequate recovery of the T790M variant using ddPCR. From a technical point of view, stabilizing buffers and adequate storage conditions are a prerequisite for translation of urinary cfDNA diagnostics into clinical routine.

Performance evaluation of fully automated cobas® 6800 CMV PCR for the detection and quantification of cytomegalovirus DNA in neonatal urine and saliva, and adult urine, saliva, and vaginal secretion.

Laboratory testing for cytomegalovirus (CMV) in bodily fluids is essential to manage congenital and prenatal CMV infection. The rapid and fully automated cobas® CMV PCR is approved only for the testing of plasma in transplant patients. To evaluate the performance of the cobas® CMV to detect and quantify CMV DNA in neonatal and adult female urine, saliva, and vaginal secretion, the limit of detection (LoD), limit of quantification (LoQ), imprecision, linearity, PCR efficiency, bias, analytical specificity, cross-reactivity, and cross-contamination of the cobas® CMV for urine, saliva, and vaginal secretion was determined. The performance of the assay was evaluated prospectively with two laboratory-developed PCR assays using neonatal and adult urine, saliva swabs, and vaginal swabs. The LoD and LoQ were 31 and 100 IU/mL, respectively, for urine, and 81 and 100 IU/mL, respectively, for vaginal secretion. The LoD and LoQ for saliva were the same (200 IU/mL). The cobas® CMV was precise (coefficient of variation ≤10%), linear (R2 ≥ 0.995), and efficient (1.07 and 1.09) between 100 and 250,000 IU/mL for the sample types. The bias and analytical specificity was <±0.30 log10 IU/mL and 100%, respectively. Cross-reactivity with non-CMV pathogens was not detected. Cross-contamination rate was 0.28%. The diagnostic accuracy, sensitivity, and specificity of the cobas® CMV for neonatal urine and saliva were ≥95.0%, ≥93.3%, and ≥90.4%, respectively. The overall percent agreement for adult urine, saliva, and vaginal secretion was 86.6%, 94.5%, and 89.4%, respectively. Taken together, the cobas® CMV demonstrated acceptable analytical and diagnostic performance, and is suitable for routine diagnostic laboratory investigation of CMV infection in neonates and adults.