Chronic lower urinary tract symptoms (LUTS) are characterised by persistent symptoms and elevated urinary leukocyte counts. Much evidence supports an infective aetiology in chronic LUTS, often coined chronic UTI, due to the poor performance of current gold standard tests to detect refractory forms of infection. Hence, we hypothesised that neutrophils from chronic LUTS patients may exhibit an aberrant response which prevents complete disease resolution. Freshly isolated circulating neutrophils from seven healthy women (control neutrophils) and four patients (patient neutrophils) were stimulated with commensal E. coli or pathogenic E. coli in artificial urine or pooled urine from controls (control urine) and patients (patient urine). Live imaging was performed at 37 °C for 15 h. Image sequences were analysed to assess neutrophil viability counts, morphometrics, cell activity and bacterial growth. Neutrophils from both cohorts were able to recognise a commensal E. coli from a pathogenic strain, with cell death occurring faster when stimulated with the latter. However, when stimulated with the same bacterial strain in control urine, patient neutrophils showed a higher level of activity and a delayed rate of death. Without bacterial stimulation, patient neutrophils died faster in patient urine. The delayed response and the shorter survival without bacteria exhibited by patient neutrophils may have implications in chronic LUTS patients, where their neutrophils are unable to effectively clear bacteria or survive for an adequate duration to exert their functions. Temporal differences in neutrophil response towards bacteria and dysregulation in neutrophil maturation or activation may lead to symptom persistence in chronic LUTS.

The vaginal and urinary microbiomes are closely linked, yet the role of this relationship in recurrent and chronic urinary tract infections (UTIs) remains uncertain. Research into genitourinary ecology and UTI has largely focused on acute infections and reproductive age groups, leaving a gap in understanding the role of the microbiome in recurrent and chronic cases. This review is aimed at presenting that disruptions within vaginal microbiota contribute to UTI chronicity in menopausal women, highlighting the potential for microbiome-targeted interventions in this high-risk group. A comprehensive literature review was conducted, with search terms including cystitis, urogenital, antibiotic, infection, and bladder, vaginal microbiome, vaginal ecology, topical oestrogen, atrophy, genitourinary syndrome of menopause, vulvo-vaginal atrophy (VVA), bacterial vaginosis and lactobacillus. Relevant articles were screened, critiqued, and synthesised based on key themes. Lactobacillus spp. appears to be the key component of a healthy vaginal microbiome. Decreased vaginal Lactobacillus abundance, seen with vaginal dysbioses and the menopause, correlates with an increased presence of urinary pathogens, increasing susceptibility to UTI. This review demonstrates that interventions to optimise vaginal ecology could reduce UTI burden. These approaches offer non-antibiotic treatment strategies, lowering antimicrobial resistance risk. However, studies frequently exclude those with chronic and recurrent infections, underscoring the necessity for more research targeting this group. This review highlights the link between disrupted vaginal ecology and recurrent and chronic UTI, and the need for expanded research into microbiome-targeted treatments. A paucity of studies researching recurrent and chronic UTI cohorts limits the evidence base for clinical generalisation, meaning that more focussed studies are needed to improve understanding and clinical management.

In this third episode of the Microbiologist in the Clinic series, clinicians and laboratory scientists share their perspectives about a 32 y/o female who has become antibiotic-dependent for her urinary symptoms. Despite escalating methods of antibiotic administration, the patient has persistent and recurrent "UTI" symptoms. Extensive testing has not provided guidance for her treating clinicians. The challenges of this clinical presentation are discussed with evidence for evaluation and treatment.

In this fourth episode of the Microbiologist in the Clinic series, clinicians and laboratory scientists share their perspectives about a 26-year-old microbiologist G1P0 who is 20 weeks pregnant. She is questioning her obstetrician’s recommendation to take systemic antibiotics for a positive urine culture obtained, as her research work focuses on the effect of antibiotics on young children. She hopes to avoid exposing her child to systemic antibiotics if possible.

Despite recognized sex-based differences in healthcare requirements, women continue to experience substantial disparities in treatment, diagnosis and research. This ‘gender health gap’ manifests through increased emergency wait times, dismissal of symptoms and inadequate research prioritization. Patient advocacy groups have emerged as powerful forces for change, successfully lobbying governments and raising awareness through social media. Addressing these disparities requires increased research funding, sex-specific study designs, improved medical education curricula and continued patient advocacy.

Phase imaging is gaining importance due to its applications in fields like biomedical imaging and material characterization. In biomedical applications, it can provide quantitative information missing in label-free microscopy modalities. One of the most prominent methods in phase quantification is the Transport-of-Intensity Equation (TIE). TIE often requires multiple acquisitions at different defocus distances, which is not always feasible in a clinical setting due to hardware constraints. To address this issue, we propose the use of chromatic aberrations to induce the required through-focus images with a single exposure, effectively generating a through-focus stack. Since the defocus distance induced by the aberrations is small, conventional TIE solvers are insufficient to address the resulting artifacts. We propose Zero-Mean Diffusion, a modified version of diffusion models designed for quantitative image prediction, and train it with synthetic data to ensure robust phase retrieval. Our contributions offer an alternative TIE approach that leverages chromatic aberrations, achieving accurate single-exposure phase measurement with white light and thus improving the efficiency of phase imaging. Additionally, we present a new class of diffusion models that are well-suited for quantitative data and have a sound theoretical basis. To validate our approach, we employ a widespread brightfield microscope equipped with a commercially available color camera. We apply our model to clinical microscopy of patients' urine, obtaining accurate phase measurements.

Chronic urinary tract infection (UTI) presents with protracted lower urinary tract symptoms and elevated urinary leukocyte counts, but its bacterial etiological agents remain obscure. In this cross-sectional investigation, we aimed to unravel the role of the bladder microbiota in chronic UTI pathogenesis by studying the host immune response. Urine samples were collected from healthy controls (HT), chronic UTI patients who had not initiated treatment (PT) and those undergoing treatment (OT), then sorted into white blood cell (WBC) and epithelial cell (EPC) fractions. Bacteria associated with both fractions were identified by chromogenic agar culture coupled with mass spectrometry and 16S rRNA sequencing. Distinct WBC-exclusive bacteria were observed in the healthy population, but this pattern was less obvious in patients, plausibly due to epithelial shedding and breaching of the urothelial barrier. We also described a bacterial fingerprint guided by Escherichia that was able to stratify patients based on symptom severity. Clustering analyses of mean rank changes revealed highly statistically significant upward and downward ecological shifts in communities of bacteria between the healthy and diseased populations. Interestingly, many of the most abundant genera identified in sequencing remained stable when compared between the study cohorts. We concluded that reshuffling of the urinary microbiome, rather than the activity of a single known urinary pathogen, could drive chronic UTI.

Inverse problems aim to determine parameters from observations, a crucial task in engineering and science. Lately, generative models, especially diffusion models, have gained popularity in this area for their ability to produce realistic solutions and their good mathematical properties. Despite their success, an important drawback of diffusion models is their sensitivity to the choice of variance schedule, which controls the dynamics of the diffusion process. Fine-tuning this schedule for specific applications is crucial but time-costly and does not guarantee an optimal result. We propose a novel approach for learning the schedule as part of the training process. Our method supports probabilistic conditioning on data, provides high-quality solutions, and is flexible, proving able to adapt to different applications with minimum overhead. This approach is tested in two unrelated inverse problems: super-resolution microscopy and quantitative phase imaging, yielding comparable or superior results to previous methods and fine-tuned diffusion models. We conclude that fine-tuning the schedule by experimentation should be avoided because it can be learned during training in a stable way that yields better results.

Urinary tract infection (UTI) is a common disorder. Its diagnosis can be made by microscopic examination of voided urine for markers of infection. This manual technique is technically difficult, time-consuming and prone to inter-observer errors. The application of computer vision to this domain has been slow due to the lack of a clinical image dataset from UTI patients. We present an open dataset containing 300 images and 3,562 manually annotated urinary cells labelled into seven classes of clinically significant cell types. It is an enriched dataset acquired from the unstained and untreated urine of patients with symptomatic UTI using a simple imaging system. We demonstrate that this dataset can be used to train a Patch U-Net, a novel deep learning architecture with a random patch generator to recognise urinary cells. Our hope is, with this dataset, UTI diagnosis will be made possible in nearly all clinical settings by using a simple imaging system which leverages advanced machine learning techniques.

In this second episode of the Microbiologist in the Clinic series, clinicians and laboratory scientists share their perspectives about a 75-year-old woman who was diagnosed with asymptomatic bacteriuria based on positive urine cultures. The patient and her GP are concerned about this laboratory finding as the patient will become immunosuppressed with planned chemotherapy. The patient has had an overactive bladder (OAB) for approximately 20 years, with good control of her urinary urgency and frequency (no incontinence) with a stable dose of OAB medication. The challenges of this clinical presentation are discussed, with evidence for evaluation and treatment.

Diagnosis and treatment of urinary tract infections (UTIs) remains stagnant. The presumption that a patient either has a UTI or does not (binary choice) is inappropriately simplistic. Laboratory diagnostic tests have not advanced for decades. The goal of UTI treatment has not been rigorously defined and may increase the prescription of potentially harmful, inappropriate antibiotics. Despite the high incidence of UTI diagnoses, the high cost of UTI treatment, and increasing concerns associated with antimicrobial resistance, the development of novel and more accurate UTI tests has not been considered a priority, in part due to the general perception that current UTI care is already sufficient. In this review, we discuss the importance of improving UTI diagnostic testing to improve treatment outcomes. We discuss the problems associated with UTI diagnosis. Urinary microbes are alive and exist in both healthy and symptomatic individuals—urine is not sterile. We specifically outline the limitations of standard urine culture methods used by clinical microbiology laboratories, explaining clearly why such methods cannot be considered to be the “gold standard,” as standard culture methods underreport most of the urinary tract microbes, including some acknowledged and many emerging uropathogens. We do not recommend abandonment of this test, as no universally accepted substitute yet exists. However, we strongly encourage the development of new and improved diagnostic tests that can both improve outcomes and preserve antibiotic stewardship.

In this first episode of the Microbiologist in the Clinic series, clinicians and laboratory scientists share their perspectives about a 30 y/o woman, who is seeking specialty consultation for frequent episodes of urinary urgency, frequency, and dysuria, which respond to short courses of antibiotics. Although her home dipsticks suggest that she has a UTI, and her urinalysis typically has a moderate number of white blood cells, her urine cultures are always negative. The challenges of this clinical presentation are discussed with evidence for evaluation and treatment.

Urinary tract infections (UTIs) exert a significant health and economic cost globally. Approximately one in four people with a previous history of UTI continue to develop recurrent or chronic infections. Research on UTI has primarily concentrated on pathogen behavior, with the focus gradually shifting to encompass the host immune response. However, these are centered on mouse models of Escherichia coli infection, which may not fully recapitulate the infective etiology and immune responses seen in humans. The emerging field of the urobiome also inadvertently confounds the discrimination of true UTI-causing pathogens from commensals. This review aims to present a novel perspective on chronic UTI by linking microbiology with immunology, which is commonly divergent in this field of research. It also describes the challenges in understanding chronic UTI pathogenesis and the human bladder immune response, largely conjectured from murine studies. Lastly, it outlines the shortcomings of current diagnostic methods in identifying individuals with chronic UTI and consequently treating them, potentially aggravating their disease due to mismanagement of prior episodes. This discourse highlights the need to consider these knowledge gaps and encourages more relevant studies of UTIs in humans.

OBJECTIVES:Granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) are autoimmune vasculitides associated with antineutrophil cytoplasm antibodies that target proteinase 3 (PR3) or myeloperoxidase (MPO) found within neutrophils and monocytes. Granulomas are exclusively found in GPA and form around multinucleated giant cells (MGCs), at sites of microabscesses, containing apoptotic and necrotic neutrophils. Since patients with GPA have augmented neutrophil PR3 expression, and PR3-expressing apoptotic cells frustrate macrophage phagocytosis and cellular clearance, we investigated the role of PR3 in stimulating giant cell and granuloma formation. METHODS:We stimulated purified monocytes and whole peripheral blood mononuclear cells (PBMCs) from patients with GPA, patients with MPA or healthy controls with PR3 or MPO and visualised MGC and granuloma-like structure formation using light, confocal and electron microscopy, as well as measuring the cell cytokine production. We investigated the expression of PR3 binding partners on monocytes and tested the impact of their inhibition. Finally, we injected zebrafish with PR3 and characterised granuloma formation in a novel animal model. RESULTS:In vitro, PR3 promoted monocyte-derived MGC formation using cells from patients with GPA but not from patients with MPA, and this was dependent on soluble interleukin 6 (IL-6), as well as monocyte MAC-1 and protease-activated receptor-2, found to be overexpressed in the cells of patients with GPA. PBMCs stimulated by PR3 formed granuloma-like structures with central MGC surrounded by T cells. This effect of PR3 was confirmed in vivo using zebrafish and was inhibited by niclosamide, a IL-6-STAT3 pathway inhibitor. CONCLUSIONS:These data provide a mechanistic basis for granuloma formation in GPA and a rationale for novel therapeutic approaches.

Ischemia is a major cause of kidney damage. Proximal tubular epithelial cells (PTECs) are highly susceptible to ischemic insults that frequently cause acute kidney injury (AKI), a potentially life-threatening condition with high mortality. Accumulating evidence has identified altered mitochondrial function as a central pathologic feature of AKI. The mitochondrial NAD + -dependent enzyme sirtuin 5 (SIRT5) is a key regulator of mitochondrial form and function, but its role in ischemic renal injury (IRI) is unknown. SIRT5 expression was increased in murine PTECs after IRI in vivo and in human PTECs (hPTECs) exposed to an oxygen/nutrient deprivation (OND) model of IRI in vitro. SIRT5-depletion impaired ATP production, reduced mitochondrial membrane potential, and provoked mitochondrial fragmentation in hPTECs. Moreover, SIRT5 RNAi exacerbated OND-induced mitochondrial bioenergetic dysfunction and swelling, and increased degradation by mitophagy. These findings suggest SIRT5 is required for normal mitochondrial function in hPTECs and indicate a potentially important role for the enzyme in the regulation of mitochondrial biology in ischemia.