Vaginal erbium laser treatment for stress urinary incontinence: A multicenter randomized sham-controlled clinical trial.

OBJECTIVE: To evaluate the efficacy and safety of non-ablative vaginal Er:YAG laser device in stress urinary incontinence (SUI) treatment. METHODS: We conducted a multicenter blinded randomized sham-controlled trial in which women with urodynamic SUI were randomization to active arm using Er:YAG laser therapy, and sham arm using sham handpiece. Patients received two treatments 1 month apart. The primary outcomes measure was 1 h pad weight test measured at 6 months. Secondary outcomes were durability of treatment success at 12 months, and questionnaires for assessment of SUI severity (ICIQ-UI SF), sexual function (PISQ-12) and HRQoL (KHQ), and incidence and severity of device related adverse events and pain (VAS). RESULTS: A total of 110 participants with SUI were recruited; 73 in the active arm and 37 in the sham arm. Two participants were excluded; one was assigned the wrong treatment and one withdrew their consent. Treatment success was observed in 36% of the sham arm and 59% of the active arm; in the latter, odds of achieving treatment success were more than three-fold higher (OR 3.63, 95% CI: 1.3-11.2, P = 0.02). HRQoL by KHQ showed significant improvement in the active versus the sham arm (OR 0.36, 95% CI: 0.15-0.87, P = 0.003). Similarly, subjective patient assessment of general and sexual function improvement with PISQ-12 and PGI-I showed superior effect over sham (OR 2.8, 95% CI: 1.2-7.0, P = 0.02 and OR 0.13, 95% CI: 0.05-0.36, P < 0.001, respectively). CONCLUSION: Non-ablative vaginal Er:YAG laser therapy significantly improves SUI symptoms versus sham treatment. Er:YAG laser therapy should be considered as a non-surgical treatment option for SUI patients.

The Women's Health Initiative cancer survivorship clinic incorporating electronic patient-reported outcomes: a study protocol for the Linking You to Support and Advice (LYSA) randomized controlled trial.

BACKGROUND: The improved survival rate for many cancers in high-income countries demands a coordinated multidisciplinary approach to survivorship care and service provision to ensure optimal patient outcomes and quality of life. This study assesses the feasibility of introducing a Women's Health Initiative cancer survivorship clinic in Ireland. METHODS: The trial https://spcare.bmj.com/content/9/2/209.short comprises an intervention and control arm. Two hundred participants will be recruited. Key eligibility (1) women with early-stage hormone receptor-positive breast or gynecologic cancer (cervix or endometrial), within 12 months of completion of primary curative therapy, and (2) access to the Internet. The complex intervention comprises a nurse-led clinic targeting symptom management through a trigger alert system, utilizing electronic patient-reported outcome (ePRO) assessments at baseline, and 2, 4, 6, 8, 10, and 12 months. It also includes input from a dietitian monitoring diet and nutritional status. The control group will receive their usual care pathway standard of care and attend the cancer survivorship clinic and complete ePRO assessments at the start and end of the study. The primary endpoint (feasibility) includes the proportion of enrolled participants who complete baseline and follow-up ePRO surveys and partake in health professional consultations after ePRO data triggers. Secondary endpoints include changes in cancer-related symptom scores assessed by ePROs, health-related Quality of Life Questionnaire (QLQ) scores, Appraisal Self-Care Agency-R scores, and adjuvant endocrine therapy medication adherence. A process evaluation will capture the experiences of participation in the study, and the healthcare costs will be examined as part of the economic analysis. Ethical approval was granted in December 2020, with accrual commencing in March 2021. DISCUSSION: This protocol describes the implementation of a parallel arm randomized controlled trial (RCT) which examines the feasibility of delivering a Cancer Survivorship Clinic. The ePRO is an innovative symptom monitoring system which detects the treatment-related effects and provides individualized support for cancer survivors. The findings will provide direction for the implementation of future survivorship care. TRIAL REGISTRATION: ClinicalTrials.gov , NCT05035173 . Retrospectively registered on September 5, 2021.

The effect of dietary phosphorus load and food matrix on postprandial serum phosphate in hemodialysis patients: a pilot study.

Background: Potential dietary strategies for controlling hyperphosphataemia include the use of protein sources with lower phosphorus bioavailability such as pulses and nuts, focus on phosphorus to protein ratios and the avoidance of all phosphate additives. Methods: We conducted a controlled crossover feeding study in 8 haemodialysis (HD) patients to investigate the acute postprandial effect of a modified versus standard low phosphorus diet for one day on serum phosphate, potassium and intact parathyroid levels in prevalent HD patients. Each participant consumed the modified diet on one day and the standard diet on a second day one week apart. The modified diet included beef and less dairy, with a lower phosphorus to protein ratio, as well as plant-based protein, whole grains, pulses and nuts containing phytates which reduces phosphorus bioavailability. Both diets were tailored for each participant to provide 1.1g protein/kg ideal body weight. Participants provided fasting bloods before breakfast, a pre-prandial sample before the lunch time main meal and samples at one-hour intervals for the four hours after the lunch time main meal, for analysis of phosphate, potassium and intact parathyroid hormone (iPTH). Results: At four hours post the lunch time main meal on each study day, individuals on the modified diet had serum phosphate readings 0.30 mmol/l lower than when on the standard diet (p-value = 0.015, 95% confidence interval [CI] -0.57, -0.04). The corresponding change in serum potassium at four hours was a decrease of 0.675 mmol/l (p-value = 0.011, CI -1.25, -0.10). Conclusions: Decreases in both serum phosphate and serum potassium readings on a modified low phosphorus diet encourage further larger studies to explore the possibility of greater food choice and healthier plant-based diets in HD patients.  ClinicalTrials.gov registration: NCT04845724 (15/04/2021).

Developing an open educational resource for open research: Protocol for the PaPOR TRAIL project.

Background: Open research involves actions at all stages of the research cycle to make the research process and outputs more transparent and accessible. Though a number of initiatives exist for researchers at PhD, post-doctoral and more senior levels, there remains a critical need for educational resources for research students at earlier career stages and across disciplines. The aim of the Principles and Practices of Open Research: Teaching, Research, Impact, and Learning (PaPOR TRaIL) project is to develop an open educational resource (OER) on the principles and practice of open research for undergraduate and master's students. Methods: In stage 1, interviews and surveys of students and supervisors are being conducted to explore students' and supervisors' knowledge, attitudes, and experiences of open research, in addition to needs and preferences for the content and delivery of the OER. Stage 2 involves development of the OER content and delivery, based on Stage 1 engagement and national and international guidance on best practice in conducting and teaching open research. In Stage 3, students and supervisors will evaluate the developed OER and provide feedback in terms of OER usability, learning experience and learning outcomes. This feedback will guide revisions and finalisation of the OER content, format and learning activities. Discussion: The PaPOR TRaIL project will develop an evidence-based OER that provides a foundation in all aspects of open research theory & practice. Teaching undergraduate and master's students open research will promote development of core research values and equip them with transferable competencies and skills, including how to conduct and use research in a trustworthy and ethical manner within and beyond academia. Enhancing teaching and learning of open research will promote better teaching and research outcomes that will benefit individuals, universities, and science more broadly.

Pilot Randomized Controlled Trial of a Standard Versus a Modified Low-Phosphorus Diet in Hemodialysis Patients.

INTRODUCTION: The standard low-phosphorus diet restricts pulses, nuts, and whole grains and other high phosphorus foods to control hyperphosphatemia. We conducted a randomized controlled trial to evaluate the effectiveness, safety, and tolerability of the modified diet, which introduced some pulses and nuts, increased the use of whole grains, increased focus on the avoidance of phosphate additives, and introduced the prescription of low-biological-value protein such as bread. METHODS: We conducted a multicenter, pragmatic, parallel-arm, open-label, randomized controlled trial of modified versus standard diet in 74 adults on hemodialysis with hyperphosphatemia over 1 month. Biochemistry was assessed using monthly laboratory tests. Dietary intake was assessed using a 2-day record of weighed intake of food, and tolerability was assessed using a patient questionnaire. RESULTS: There was no significant difference in the change in serum phosphate between the standard and modified diets. Although total dietary phosphorus intake was similar, phytate-bound phosphorus, found in pulses, nuts, and whole grains, was significantly higher in the modified diet (P < 0.001). Dietary fiber intake was also significantly higher (P < 0.003), as was the percentage of patients reporting an increase in bowel movements while following the modified diet (P = 0.008). There was no significant difference in the change in serum potassium or in reported protein intake between the 2 diets. Both diets were similarly well tolerated. CONCLUSION: The modified low phosphorus diet was well tolerated and was associated with similar phosphate and potassium control but with a wider food choice and greater fiber intake than the standard diet.

Reverse epitope mapping of the E2 glycoprotein in antibody associated hepatitis C virus.

The humoral immune system responds to chronic hepatitis C virus (HCV) infection by producing neutralising antibodies (nAb). In this study we generated three HCV pseudoparticles in which E1E2 glycoprotein sequence was targeted by the host humoral immune system. We used patient derived virus free Fabs (VF-Fabs) obtained from HCV genotype 1a (n = 3), genotype 1b (n = 7) and genotype 3a (n = 1) for neutralisation of HCVpp produced in this study both individually and in combination. Based on the available anti-HCV monoclonal nAb mapping information we selected amino acid region 384-619 for conformational epitope mapping. Amongst our notable findings, we observed significant reduction in HCVpp infectivity (p<0.05) when challenged with a combination of inter genotype and subtype VF-Fabs. We also identified five binding motifs targeted by patient derived VF-Fab upon peptide mapping, of which two shared the residues with previously reported epitopes. One epitope lies within an immunodominant HVR1 and two were novel. In summary, we used a reverse epitope mapping strategy to identify preferred epitopes by the host humoral immune system. Additionally, we have combined different VF-Fabs to further reduce the HCVpp infectivity. Our data indicates that combining the antigen specificity of antibodies may be a useful strategy to reduce (in-vitro) infectivity.

Humoral immune system targets clonotypic antibody-associated hepatitis C virus.

Hypervariable region 1 (HVR1) is one of the potential neutralization domains in the E2 glycoprotein of hepatitis C virus (HCV). Point mutations of the HVR1 can lead to humoral immune escape in HCV-infected patients. In this study, we segregated the chronically infected viraemic sera from HCV-infected patients into populations of antibody-free virus and antibody-associated virus (AAV) and mapped potential epitopes within the E1E2 gene junction of AAV sequences (residues 364-430). Furthermore, we generated HCV pseudoparticles (HCVpp) derived from AAV sequences to assess their infectivity. We studied the neutralization potential of virus-free Fab obtained from antibody-virus complexes, in the HCVpp system. We observed selective targeting of clonotypic HCV variants from the quasispecies pool. Moreover, we identified potential neutralizing epitopes within the HVR1 and an additional epitope that overlapped with a broadly neutralizing AP33 epitope (amino acid 412-423 in E2). We observed a marked difference in the infectivity of HCVpp generated using E1E2 sequences isolated from AAV. We document reduction in the infectivity of HCVpp-H77 and HCVpp derived from AAV sequences when challenged with virus-free Fab. Our results provide novel insights into the complexities of engagement between HCV and the humoral immune system.

Ultradeep Pyrosequencing of Hepatitis C Virus to Define Evolutionary Phenotypes.

Analysis of hypervariable regions (HVR) using pyrosequencing techniques is hampered by the ability of error correction algorithms to account for the heterogeneity of the variants present. Analysis of between-sample fluctuations to virome sub-populations, and detection of low frequency variants, are unreliable through the application of arbitrary frequency cut offs. Cumulatively this leads to an underestimation of genetic diversity. In the following technique we describe the analysis of Hepatitis C virus (HCV) HVR1 which includes the E1/E2 glycoprotein gene junction. This procedure describes the evolution of HCV in a treatment naïve environment, from 10 samples collected over 10 years, using ultradeep pyrosequencing (UDPS) performed on the Roche GS FLX titanium platform ( Palmer et al., 2014 ). Initial clonal analysis of serum samples was used to inform downstream error correction algorithms that allowed for a greater sequence depth to be reached. PCR amplification of this region has been tested for HCV genotypes 1, 2, 3 and 4.

Synonymous Co-Variation across the E1/E2 Gene Junction of Hepatitis C Virus Defines Virion Fitness.

Hepatitis C virus is a positive-sense single-stranded RNA virus. The gene junction partitioning the viral glycoproteins E1 and E2 displays concurrent sequence evolution with the 3'-end of E1 highly conserved and the 5'-end of E2 highly heterogeneous. This gene junction is also believed to contain structured RNA elements, with a growing body of evidence suggesting that such structures can act as an additional level of viral replication and transcriptional control. We have previously used ultradeep pyrosequencing to analyze an amplicon library spanning the E1/E2 gene junction from a treatment naïve patient where samples were collected over 10 years of chronic HCV infection. During this timeframe maintenance of an in-frame insertion, recombination and humoral immune targeting of discrete virus sub-populations was reported. In the current study, we present evidence of epistatic evolution across the E1/E2 gene junction and observe the development of co-varying networks of codons set against a background of a complex virome with periodic shifts in population dominance. Overtime, the number of codons actively mutating decreases for all virus groupings. We identify strong synonymous co-variation between codon sites in a group of sequences harbouring a 3 bp in-frame insertion and propose that synonymous mutation acts to stabilize the RNA structural backbone.

A single amino acid change in the hypervariable region 1 of hepatitis C virus genotype 4a aids humoral immune escape.

Longitudinal analysis of chronic hepatitis C virus (HCV) infection has shown that the virus has several adaptive strategies that maintain persistence and infectivity over time. We examined four serum samples from the same chronically infected HCV genotype 4a patient for the presence of IgG antibody-associated virus. RNA was isolated from antibody-associated and antibody-free virions. Subsequent to sequence analysis, 27 aa hypervariable region 1 (HVR1) peptides were used to test the humoral immune escape. We demonstrated that differential peptide binding of Fab was associated with a single amino acid change. We provide direct evidence of natural humoral immune escape by HCV within HVR1.

Network Analysis of the Chronic Hepatitis C Virome Defines Hypervariable Region 1 Evolutionary Phenotypes in the Context of Humoral Immune Responses.

UNLABELLED: Hypervariable region 1 (HVR1) of hepatitis C virus (HCV) comprises the first 27 N-terminal amino acid residues of E2. It is classically seen as the most heterogeneous region of the HCV genome. In this study, we assessed HVR1 evolution by using ultradeep pyrosequencing for a cohort of treatment-naive, chronically infected patients over a short, 16-week period. Organization of the sequence set into connected components that represented single nucleotide substitution events revealed a network dominated by highly connected, centrally positioned master sequences. HVR1 phenotypes were observed to be under strong purifying (stationary) and strong positive (antigenic drift) selection pressures, which were coincident with advancing patient age and cirrhosis of the liver. It followed that stationary viromes were dominated by a single HVR1 variant surrounded by minor variants comprised from conservative single amino acid substitution events. We present evidence to suggest that neutralization antibody efficacy was diminished for stationary-virome HVR1 variants. Our results identify the HVR1 network structure during chronic infection as the preferential dominance of a single variant within a narrow sequence space. IMPORTANCE: HCV infection is often asymptomatic, and chronic infection is generally well established in advance of initial diagnosis and subsequent treatment. HVR1 can undergo rapid sequence evolution during acute infection, and the variant pool is typically seen to diverge away from ancestral sequences as infection progresses from the acute to the chronic phase. In this report, we describe HVR1 viromes in chronically infected patients that are defined by a dominant epitope located centrally within a narrow variant pool. Our findings suggest that weakened humoral immune activity, as a consequence of persistent chronic infection, allows for the acquisition and maintenance of host-specific adaptive mutations at HVR1 that reflect virus fitness.

Analysis of the evolution and structure of a complex intrahost viral population in chronic hepatitis C virus mapped by ultradeep pyrosequencing.

UNLABELLED: Hepatitis C virus (HCV) causes chronic infection in up to 50% to 80% of infected individuals. Hypervariable region 1 (HVR1) variability is frequently studied to gain an insight into the mechanisms of HCV adaptation during chronic infection, but the changes to and persistence of HCV subpopulations during intrahost evolution are poorly understood. In this study, we used ultradeep pyrosequencing (UDPS) to map the viral heterogeneity of a single patient over 9.6 years of chronic HCV genotype 4a infection. Informed error correction of the raw UDPS data was performed using a temporally matched clonal data set. The resultant data set reported the detection of low-frequency recombinants throughout the study period, implying that recombination is an active mechanism through which HCV can explore novel sequence space. The data indicate that polyvirus infection of hepatocytes has occurred but that the fitness quotients of recombinant daughter virions are too low for the daughter virions to compete against the parental genomes. The subpopulations of parental genomes contributing to the recombination events highlighted a dynamic virome where subpopulations of variants are in competition. In addition, we provide direct evidence that demonstrates the growth of subdominant populations to dominance in the absence of a detectable humoral response. IMPORTANCE: Analysis of ultradeep pyrosequencing data sets derived from virus amplicons frequently relies on software tools that are not optimized for amplicon analysis, assume random incorporation of sequencing errors, and are focused on achieving higher specificity at the expense of sensitivity. Such analysis is further complicated by the presence of hypervariable regions. In this study, we made use of a temporally matched reference sequence data set to inform error correction algorithms. Using this methodology, we were able to (i) detect multiple instances of hepatitis C virus intrasubtype recombination at the E1/E2 junction (a phenomenon rarely reported in the literature) and (ii) interrogate the longitudinal quasispecies complexity of the virome. Parallel to the UDPS, isolation of IgG-bound virions was found to coincide with the collapse of specific viral subpopulations.

Insertion and recombination events at hypervariable region 1 over 9.6 years of hepatitis C virus chronic infection.

Hepatitis C virus (HCV) exists as a quasispecies within an infected individual. We have previously reported an in-frame 3 bp insertion event at the N-terminal region of the E2 glycoprotein from a genotype 4a HCV isolate giving rise to an atypical 28 aa hypervariable region (HVR) 1. To further explore quasispecies evolution at the HVR1, serum samples collected over 9.6 years from the same chronically infected, treatment naïve individual were subjected to retrospective clonal analysis. Uniquely, we observed that isolates containing this atypical HVR1 not only persisted for 7.6 years, but dominated the quasispecies swarm. Just as striking was the collapse of this population of variants towards the end of the sampling period in synchrony with variants containing a classical HVR1 from the same lineage. The replication space was subsequently occupied by a second minor lineage, which itself was only intermittently detectable at earlier sampling points. In conjunction with the observed genetic shift, the coexistence of two distinct HVR1 populations facilitated the detection of putative intra-subtype recombinants, which included the identification of the likely ancestral parental donors. Juxtaposed to the considerable plasticity of the HVR1, we also document a degree of mutational inflexibility as each of the HVR1 subpopulations within our dataset exhibited overall genetic conservation and convergence. Finally, we raise the issue of genetic analysis in the context of mixed lineage infections.

The pan-genotype specificity of the hepatitis C virus anti-core monoclonal antibody C7-50 is contingent on the quasispecies profile of a population.

The inter/intra-genotype quasispecies makeup of hepatitis C virus (HCV) has retarded the development of antibodies capable of pan-genotype reactivity. Mutations, even in conserved domains, are tolerated to a degree. In this report, we characterise the pan-genotype specificity of the commercially available monoclonal anti-HCV core antibody C7-50. We demonstrate the antibody's ability to detect HCV core protein following infection of Huh7 cells with serum-derived HCV of genotypes 2-5 and that a single-site polymorphism in a genotype 3a core amino acid sequence is sufficient to disrupt antibody recognition of the epitope. This same polymorphism is a feature of genotype 3 viruses.