Sacral neuromodulation device biofilm differs in the absence and presence of infection, harbors antibiotic resistance genes, and is reproducible in vitro.

INTRODUCTION/PURPOSE: Sacral neuromodulation (SNM) is effective therapy for overactive bladder refractory to oral therapies, and non-obstructive urinary retention. A subset of SNM devices is associated with infection requiring surgical removal. We sought to compare microbial compositions of explanted devices in the presence and absence of infection, by testing phase, and other clinical factors, and to investigate antibiotic resistance genes present in the biofilms. We analyzed resistance genes to antibiotics used in commercially-available anti-infective device coating/pouch formulations. We further sought to assess biofilm reconstitution by material type and microbial strain in vitro using a continuous-flow stir tank bioreactor, which mimics human tissue with an indwelling device. We hypothesized that SNM device biofilms would differ in composition by infection status, and genes encoding resistance to rifampin and minocycline would be frequently detected. MATERIALS/METHODS: Patients scheduled to undergo removal or revision of SNM devices were consented per IRB-approved protocol (IRB 20-415). Devices were swabbed intraoperatively upon exposure, with controls and precautions to reduce contamination of the surrounding field. Samples and controls were analyzed with next-generation sequencing and RT-PCR, metabolomics, and culture-based approaches. Associations between microbial diversity or microbial abundance, and clinical variables were then analyzed using t-tests and ANOVA. Reconstituted biofilm deposition in vitro using the bioreactor was compared by microbial strain and material type using plate-based assays and scanning electron microscopy. RESULTS: Thirty seven devices were analyzed, all of which harbored detectable microbiota. Proteobacteria, Firmicutes and Actinobacteriota were the most common phyla present overall. Beta-diversity differed in the presence versus absence of infection (p = 0.014). Total abundance, based on normalized microbial counts, differed by testing phase (p < 0.001), indication for placement (p = 0.02), diabetes mellitus (p < 0.001), cardiac disease (p = 0.008) and history of UTI (p = 0.008). Significant microbe-metabolite interaction networks were identified overall and in the absence of infection. 24% of biofilms harbored the tetA tetracycline/minocycline resistance gene and 53% harbored the rpoB rifampin resistance gene. Biofilm was reconstituted across tested strains and material types. Ceramic and titanium did not differ in biofilm deposition for any tested strain. CONCLUSIONS: All analyzed SNM devices harbored microbiota. Device biofilm composition differed in the presence and absence of infection and by testing phase. Antibiotic resistance genes including to rifampin and tetracycline/minocycline, which are used in commercially-available anti-infective pouches, were frequently detected. Isolated organisms from SNM devices demonstrated the ability to reconstitute biofilm formation in vitro. Biofilm deposition was similar between ceramic and titanium, materials used in commercially-available SNM device casings. The findings and techniques used in this study together provide the basis for the investigation of the next generation of device materials and coatings, which may employ novel alternatives to traditional antibiotics. Such alternatives might include bacterial competition, quorum-sensing modulation, or antiseptic application, which could reduce infection risk without significantly selecting for antibiotic resistance.

How do we make progress in phenotyping patients with LUT such as OAB and underactive detrusor, including using urine markers and microbiome data, in order to personalize therapy? ICI-RS 2023: Part 1.

INTRODUCTION: Overactive bladder (OAB) and Underactive bladder (UAB) could be associated with metabolic syndrome, affective disorders, sex hormone deficiency, changes in urinary microbiota, functional gastrointestinal disorders, or autonomic nervous system dysfunction. OBJECTIVES: The aim of this Think Tank was to provide a guide on how to investigate OAB and/or detrusor underactivity (DU) patients to better clarify the underlying pathophysiology and possibly personalize the treatment. METHODS: A compendium of discussion based on the current evidence related to phenotyping patients with OAB or DU investigating metabolic, neurogical, psychological and gastrointestinal aspects with the aim to personalize the treatment. RESULTS AND CONCLUSIONS: The article emphasizes the critical significance of adopting a comprehensive yet tailored approach to phenotyping patients with lower urinary tract symptoms, such as OAB and UAB. The intricate interplay between the lower urinary tract and various factors, metabolic, neurological, psychological, and gastrointestinal can define unique LUT profiles, enabling personalized therapies to replace the one-size-fits-all approach.

How do we make progress in phenotyping patients with lower urinary tract such as overactive bladder and underactive detrusor, including using urine markers and microbiome data, to personalize therapy? ICI-RS 2023-Part 2.

INTRODUCTION: Overactive bladder (OAB) and underactive bladder (UAB) could be associated with metabolic syndrome, affective disorders, sex hormone deficiency, changes in urinary microbiota, functional gastrointestinal disorders, or autonomic nervous system dysfunction. OBJECTIVES: The aim of this Think Tank was to provide a guide on how to investigate OAB and/or detrusor underactivity (DU) patients to better clarify the underlying pathophysiology and possibly personalize the treatment. METHODS: A compendium of discussion based on the current evidence related to phenotyping patients with OAB or DU using urodynamic tests, functional neuro-imaging, urinary markers, and microbiome. RESULTS AND CONCLUSIONS: The article emphasizes the critical significance of adopting a comprehensive yet tailored approach to phenotyping patients with lower urinary tract (LUT) symptoms, such as OAB and UAB. The intricate interplay between the LUT and various factors, metabolic, neurological, psychological, and gastrointestinal can define unique LUT profiles, enabling personalized therapies to replace the one-size-fits-all approach.

Interplay between bladder microbiota and overactive bladder symptom severity: a cross-sectional study.

BACKGROUND: It is widely accepted that there exist microbiota communities in urinary tract of healthy individuals. Imbalance in the urinary microbiome plays important roles in the development of various benign urological conditions including lower urinary track symptoms (LUTS) and overactive bladder (OAB). However, whether alteration in urinary microbiome exerts influence on the severity of OAB symptom has yet to be elucidated. The purpose of this study is to examine the correlation between urinary microbiome and the severity of OAB. METHODS: A total of 70 OAB patients were recruited to finish overactive bladder symptom score (OABSS) questionnaires. Catheterized urine samples were obtained for 16S rRNA gene sequencing. The species richness and evenness were evaluated by α diversity, and the difference in urinary microbiome between patients with mild or moderate/severe severity was evaluated by ß diversity. The relationship between urinary microbiome and the severity of OAB symptom was evaluated using Pearson's correlation analysis. RESULTS: Mild patients (OABSS ≤ 5, n = 17) had lower bacterial diversity (Simpson index, P = 0.024) and richness (Chao1, P = 0.023) than those with moderate/severe symptom (OABSS > 5, n = 53). Beta-diversity of urinary microbiome between two groups were significantly different. Furthermore, the score of OABSS was positively correlated with the richness index (Chao1, P = 0.002) and diversity index (Shannon index, P = 0.044) of urinary microbiome. Certain bacterial genera (e.g., Porphyromona and Prevotella) were significantly correlated with severity of OAB sub-symptoms. CONCLUSION: This study demonstrated that urinary microbiome was intimately correlated with the severity of OAB symptom and the increase of the diversity and richness of urinary microbiome was accompanied by more severe OAB symptoms, indicating that urinary dysbiosis may play pivotal roles in the deterioration of functional bladder diseases.

The gut, vaginal, and urine microbiome in overactive bladder: a systematic review.

INTRODUCTION AND HYPOTHESIS: The objective was to systemically review the current literature on the association of gut, vaginal, and urinary dysbiosis in female patients with overactive bladder (OAB). METHODS: We performed a comprehensive literature search following the Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) protocols for systematic reviews. In the EMBASE, CINAHL, and Medline databases, a search was conducted using key words such as "microbiome," "microbiota," "microflora," "overactive bladder," "urge," "gut," "vaginal." Articles were screened using the online tool www.covidence.org . Two independent reviewers screened studies at each stage and resolved conflicts together. We excluded papers that discussed pediatric patients and animal studies. In total, 13 articles met this criterion, which included 6 abstracts. RESULTS: After identifying 817 unique references, 13 articles met the criteria for data extraction. Articles were published from 2017 to 2021. No study reported the same microbiota abundance, even in healthy individuals. Overall, there was a loss of bacterial diversity in OAB patients compared with controls. Additionally, the bacterial composition of the controls and OAB patients was not significantly different, especially if the urine was collected midstream. Overall, the composition of the microbiome is dependent on the specimen collection methodology, and the metagenomic sequencing technique utilized. OAB urine microbiome is more predisposed to alteration from the gut or vaginal influences than in controls. CONCLUSIONS: Current evidence suggested a potential relationship among gut, vaginal, and urinary microbiome in OAB patients, but there are very limited studies.

The influence of gut microbiome on progression of overactive bladder symptoms: a community-based 3-year longitudinal study in Aomori, Japan.

PURPOSE: To assess the influence of gut microbiome on overactive bladder (OAB) symptoms progression. METHODS: This was a 3-year longitudinal study, Hirosaki in Japan. We assessed OAB symptoms and reviewed the medication records of each subject in 2016. We extracted 16S rRNA genes from fecal samples and analyzed gut microbiomes via next-generation sequencing. We evaluated the changes in urinary urgency (UU) and/or urgent urinary incontinence (UUI) from 2016 to 2019. We defined UU/UUI-progression as exacerbation of UU and/or UUI. We compared the clinical backgrounds and microbiota structure between UU/UUI-progression subjects and non-progression (controls). We assessed the impact of gut microbiome on the UU/UUI-progression via multivariate logistic regression analyses. RESULTS: Of 669 subjects, 126 were UU/UUI-progression subjects. These subjects had a higher age and prevalence of proton pump inhibitor (PPI) use (14% vs. 5.4%, P = 0.003), irritable bowel syndrome, sleep disturbance, and metabolic syndrome than those without. We found the different microbiota structures between subjects with UU/UUI-progression and those without. A higher relative abundance of genus Streptococcus (harmful bacterial genus for human health) appeared in UU/UUI-progression subjects (3.8% vs. 2.3%, P < 0.001). Multivariate analysis revealed that age ≥ 65 years, current smoking, sleep disturbance, metabolic syndrome, and genus Streptococcus (Odds ratio: 1.05, P = 0.029) were independent risk factors for UU/UUI-progression. PPI use turned to be a significant risk factor on a multivariate analysis without including genus Streptococcus. CONCLUSIONS: Gut microbiome might be associated with a risk for OAB symptoms progression. PPI use might cause gut dysbiosis and increase this risk.

Urinary Biomarkers in Overactive Bladder: Revisiting the Evidence in 2019.

CONTEXT: In overactive bladder (OAB), after an initial outbreak of research, it is more consensual that biomarkers may be better used to phenotype patients. Herein, we revisit this topic, including some of the most promising biomarkers. OBJECTIVE: To provide a comprehensive analysis of the actual role of biomarkers in OAB. EVIDENCE ACQUISITION: A PubMed-based literature search was conducted, including the most relevant articles published in the last 15 yr, on nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), adenosine triphosphate (ATP), genomics, and microbiota as OAB biomarkers. Articles with no full text available or not written in English were excluded. Additional reviews were included. EVIDENCE SYNTHESIS: Urinary NGF, BDNF, and ATP are increased in many OAB patients. These biomarkers can help identify OAB phenotypes and select the ideal candidates for new therapies directed to neurotrophic and purinergic pathways. Circulating urinary miRNA may be useful for establishing the ideal moment for bladder outlet obstruction relief and will eventually lead to the development of therapeutic agents that inhibit or reverse fibrotic pathways in the bladder. Urinary microbiota seems to be related to OAB symptoms, in particular urgency urinary incontinence, and may have strong implications in the prevention, diagnosis, and treatment of OAB. CONCLUSIONS: In the future, physicians may consider the use of biomarkers to identify distinct OAB phenotypes, with distinct causal mechanisms, selecting patients for specific target therapies with expected better outcomes. PATIENT SUMMARY: Overactive bladder biomarkers can be useful for phenotype patients and for selecting more effective target therapies.

A Comprehensive Review of Overactive Bladder Pathophysiology: On the Way to Tailored Treatment.

CONTEXT: Current literature suggests that several pathophysiological factors and mechanisms might be responsible for the nonspecific symptom complex of overactive bladder (OAB). OBJECTIVE: To provide a comprehensive analysis of the potential pathophysiology underlying detrusor overactivity (DO) and OAB. EVIDENCE ACQUISITION: A PubMed-based literature search was conducted in April 2018, to identify randomised controlled trials, prospective and retrospective series, animal model studies, and reviews. EVIDENCE SYNTHESIS: OAB is a nonspecific storage symptom complex with poorly defined pathophysiology. OAB was historically thought to be caused by DO, which was either "myogenic" (urgency initiated from autonomous contraction of the detrusor muscle) or "neurogenic" (urgency signalled from the central nervous system, which initiates a detrusor contraction). Patients with OAB are often found to not have objective evidence of DO on urodynamic studies; therefore, alternative mechanisms for the development of OAB have been postulated. Increasing evidence on the role of urothelium/suburothelium and bladder afferent signalling arose in the early 2000s, emphasising an afferent "urotheliogenic" hypothesis, namely, that urgency is initiated from the urothelium/suburothelium. The urethra has also recently been regarded as a possible afferent origin of OAB-the "urethrogenic" hypothesis. Several other pathophysiological factors have been implicated, including metabolic syndrome, affective disorders, sex hormone deficiency, urinary microbiota, gastrointestinal functional disorders, and subclinical autonomic nervous system dysfunctions. These various possible mechanisms should be considered as contributing to diagnostic and treatment algorithms. CONCLUSIONS: There is a temptation to label OAB as "idiopathic" without obvious causation, given the poorly understood nature of its pathophysiology. OAB should be seen as a complex, multifactorial symptom syndrome, resulting from multiple potential pathophysiological mechanisms. Identification of the underlying causes on an individual basis may lead to the definition of OAB phenotypes, paving the way for personalised medical care. PATIENT SUMMARY: Overactive bladder (OAB) is a storage symptom syndrome with multiple possible causes. Identification of the mechanisms causing a patient to experience OAB symptoms may help tailor treatment to individual patients and improve outcomes.

The urinary microbiome in patients with refractory urge incontinence and recurrent urinary tract infection.

INTRODUCTION AND HYPOTHESIS: Urinary urge incontinence is a chronic, debilitating condition that is difficult to treat. Patients refractory to standard antimuscarinic therapy often experience recurrent urinary tract infections (rUTIs). The microbiota of these refractory patients with rUTI remains unexplored. METHODS: A midstream urine (MSU) sample was collected from patients with refractory urge incontinence and coexistent rUTI during acute symptomatic episodes. Culture-based diagnosis was performed using routine microbiological methods. Culture-independent profiling was performed using bacterial 16S RNA profiling. E. coli strain typing was performed by amplicon pyrosequencing of the fimH gene. RESULTS: Over 2 years, 39 patients with refractory urge incontinence and coexistent rUTI were studied, yielding 9 severely affected cases. These 9 patients were carefully monitored for a further 2 years, resulting in the collection of 102 MSU samples, 70 of which were diagnosed as UTI (median of 8 UTIs/woman). Culture-independent analysis of 38 of these samples revealed the existence of a diverse urinary microbiota. Strain typing of E. coli identified instances of rUTI caused by the same persisting strain and by new infecting strains. CONCLUSIONS: Patients with refractory urge incontinence and coexistent rUTI possess a diverse urinary microbiota, suggesting that persistent bladder colonisation might augment the pathology of their chronic condition.

A blinded observational cohort study of the microbiological ecology associated with pyuria and overactive bladder symptoms.

INTRODUCTION AND HYPOTHESIS: This study sought to characterise the microbial ecology of the lower urinary tract in patients with symptoms of overactive bladder (OAB) using culture of the urinary urothelial cell sediment. The pathological significance of the microbiome was assessed through its relationship with known urothelial inflammatory markers and patient reported symptoms. METHODS: Adult female patients with OAB symptoms and asymptomatic controls were assessed at 12 study visits scheduled every 4 weeks. At each visit, all participants provided a clean-catch midstream urine (MSU) that was analysed to count white and uroepithelial cells, submitted to standard culture and spun urothelial-cell-sediment culture. Symptoms were assessed using validated questionnaires. RESULTS: This analysis shows that OAB patients differ consistently from controls, demonstrating differences in bacterial ecology (t -4.57, p 0.0001), in the microscopic pyuria count (t -6.37, p 0.0001) and presence of infected urothelial cells (t -4.21, p 0.0001). The primary outcome measure of bacterial growth [colony-forming units (CFU) ml-1] was higher in OAB patients than in controls throughout the 12 months. Data showed a correlation between symptoms and pyuria, with notable urgency correlating with pyuria and epithelial cell shedding. The routine urine cultures (with a threshold of reporting a positive result as 105 CFU/ml) were unable to distinguish OAB patients from controls. However, sediment cultures differed significantly, and there was a correlated increased immune response amongst OAB patients. CONCLUSIONS: This study supports the need to re-examine the OAB phenotype given this association with microbial colonisation.

An Integrative Review of Current Research on the Role of the Female Urinary Microbiota in Overactive Bladder Symptoms.

This integrative review explores current evidence on microbiota of the female urinary tract as it relates to overactive bladder (OAB) symptoms. Six articles were identified for review. Findings suggest a possible link between the female urinary microbiome and OAB symptom presentation.

Urinary Microbiome and Psychological Factors in Women with Overactive Bladder.

Objectives: Emerging evidence indicates that alterations to the urinary microbiome are related to lower urinary tract symptoms. Overactive bladder (OAB) is a common disorder with complex etiologies and usually accompanied by psychological diseases. More information concerning the urinary microbiome and psychological factors in OAB is required. The aim of this study was to characterize the female urinary microbiome associated with OAB and investigate the relationships between urinary microbiome and psychological factors. Methods: Thirty women with OAB and 25 asymptomatic controls were recruited and asked to finish the Overactive Bladder Symptom Score, Self-Rating Anxiety Scale and Self-Rating Depression Scale. Urine specimens were collected by transurethral catheterization and processed for 16S rRNA gene sequencing using Illumina MiSeq. Sequencing reads were processed using QIIME. LEfSe revealed significant differences in bacterial genera between controls and OAB patients. The relationships between the diversity of the urinary microbiome and psychological scores were identified by Pearson's correlation coefficient. Results: We found that bacterial diversity (Simpson index) and richness (Chao1) were lower in OAB samples compared to controls (P both = 0.038). OAB and control bacterial communities were significantly different (based on weighted UniFrac distance metric, R = 0.064, P = 0.037). LEfSe demonstrated that 7 genera were increased (e.g., Proteus and Aerococcus) and 13 were reduced (e.g., Lactobacillus and Prevotella) in OAB group compared to controls. There were negative correlations between scores on Self-Rating Depression Scale and both richness (Chao1, r = -0.458, P = 0.011) and diversity (Shannon index, r = -0.516, P = 0.003) of urinary microbiome in OAB group. Some bacterial genera of OAB women with anxiety or depression were significantly different from those without. Conclusions: The aberrant urinary microbiome with decreased diversity and richness may have strong implications in pathogenesis and treatment of OAB. Psychological conditions were correlated with characteristics of urinary microbiome in women with OAB. Further research is needed to understand the connection between central nervous system and urinary microbiome.

A case controlled study examining the bladder microbiome in women with Overactive Bladder (OAB) and healthy controls.

OBJECTIVE: To characterise the microbiome in healthy women with no bladder symptoms and to compare this to the bladder microbiome in patients with overactive bladder syndrome (OAB). STUDY DESIGN: MSU specimens from 63 women with OAB were compared to urine from 35 controls. Urine was centrifuged and the resulting sediment pellet was re-suspended in supernatant and plated under aerobic conditions for 48h and anaerobic conditions for 7days. Each morphologically distinct colony was purity plated. Bacterial colonies were lysed and polymerase chain reaction undertaken to amplify the 16s ribosomal RNA gene. This DNA was purified and sequenced allowing identification of bacterial genera. RESULTS: The mean number of different bacterial genera was 5.0 in both controls and OAB patients (p=0.99). The uropathogenic bacteria Proteus (P=0.01) was more commonly isolated from women with OAB. The genus lactobacillus was present less commonly in urine from OAB patients when compared to urine taken from controls (p=0.02). Overall the most commonly grown bacteria were staphylococcus (grown in 59% of samples), streptococccus (51%), corynebacterium (37%) and lactobacillus (28%). A total of 95 different genera were identified from the urine samples. CONCLUSION: The female human bladder has a diverse microbiome with stastistically significant differences between bacterial species present in OAB patients and controls.

Is chronic urinary infection a cause of overactive bladder?

Overactive bladder (OAB) is a diagnosis resulting from a combination of multiple underlying factors. Current traditional treatments are based on anticholinergic blockade which have marginal benefits and are associated with poor tolerability and continuation rates. There is mounting evidence that chronic low grade bacterial bladder colonisation may exacerbate OAB symptoms and may explain why the current treatment strategies are not always successful. However, standard diagnostic laboratory tests to identify the presence of such bacterial infection are unreliable. Newer technologies such as RNA sequencing and extended culture techniques, show that urine is not sterile and organisms that are found in urine may be responsible for OAB symptoms. This article aims to review the current evidence suggesting that micro-organisms in urine may be important in the aetiology of OAB or may exacerbate OAB symptoms.

Urinary tract infections in women with urogynaecological symptoms.

PURPOSE OF REVIEW: Urinary tract infections are common in the field of urogynaecology. Women with persistent urinary symptoms seem more likely to have bacteriuria despite negative cultures. In this review, we will give an overview of the recent insights on the relationship between urinary tract infection and persistent urinary symptoms and possible new therapeutic options. RECENT FINDINGS: Recently published articles evaluated the prevalence of low-count bacteriuria (≥10 CFU/ml) or intracellular bacterial communities in women with overactive bladder symptoms (OAB). Differences in urinary microbioma observed in women with and without OAB symptoms were evaluated. In the light of these findings, current screening strategies were discussed and alternative screening methods for bacteriuria developed. SUMMARY: Low-count bacteriuria (≥10 CFU/ml) seems to be more prevalent in women with OAB. Also intracellular bacterial communities are more commonly detected in these women. The microbioma found in women with urinary symptoms appeared to differ from healthy controls. The current screening methods might be insufficient as they are targeted at detecting uropathogenic Escherichia coli, mostly using a detection threshold of at least 10 CFU/ml and failing to detect intracellular bacterial communities. Studies evaluating the efficacy of treating women with low-count bacteriuria are limited but promising.

[Bacterial ecology and resistance to antibiotics in patients with neurogenic overactive bladder treated with intravesical botulinum toxin injections]. / Écologie bactérienne et profils de résistance aux antibiotiques chez les patients avec hyperactivité du détrusor d'origine neurologique, traités par injections de toxine botulique intradétrusoriennes.

INTRODUCTION: For the last ten years, botulinum neurotoxin type A has become the gold standard for the treatment of neurogenic overactive detrusor. Bacterial colonization is common for these patients using clean intermittent self-catheterization, and toxin injections are at risk of urinary tract infections. OBJECTIVES: The aim of our study was to determine the prevalence of different germs and their resistance to antibiotics in patients with neurogenic bladder, treated with intravesical botulinum toxin injections. MATERIAL AND METHOD: This epidemiologic study took place from September to October 2012 in a urodynamic and neurourology unit in a teaching hospital in Paris, France. RESULTS: Eighty patients with a valid urine culture according to our protocol, were included. Fourty-four culture were positive with 45 bacteria. We found an Escherichia coli in 42.5%, a Klebsiella pneumoniae in 7.5%, a Citrobacter freundii and an enterococcus in 2.5%, and a Staphylococcus aureus in 1.25%. Penicillin resistance were found in 51.11%, 3rd generation cephalosporins in 8.89%, quinolones in 28.89% and sulfamids in 24.44%. None were resistant to fosfomycin. CONCLUSION: E. coli was the most frequent bacterium. No resistance to fosfomycin was found.

Overactive bladder in women: does low-count bacteriuria matter? A review.

Since the description of the overactive bladder (OAB) syndrome, which excludes infection, the precise definition of significant bacteriuria in these women is critical. The traditional definition of 'significant' bacteriuria is >10(5) colony-forming units/ml which was described 50 years ago by a renal physician whose primary interest was the prevention of pyelonephritis. Subsequent studies have shown this to be an insensitive threshold in women with acute lower urinary tract symptoms. Bacterial counts between 10(2) and 10(5) CFU/ml ('low-count bacteriuria') are now considered important in women with acute dysuria and warrant treatment. However, these findings have been slow to translate into routine clinical practice. In addition, the role of low-count bacteriuria in women with OAB symptoms (frequency/urgency/nocturia) without dysuria is poorly studied. One recent study has shown low-count bacteriuria to be more prevalent among women with severe OAB than bacteriuria >10(5) CFU/ml. We present an outline of the history of this issue and summarise current microbiological and clinical concepts.