Time Required for Nanopore Whole-Genome Sequencing of Neisseria gonorrhoeae for Identification of Phylogenetic Relationships.

BACKGROUND: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global health challenge. Limitations to AMR surveillance reporting, alongside reduction in culture-based susceptibility testing, has resulted in a need for rapid diagnostics and strain detection. We investigated Nanopore sequencing time, and depth, to accurately identify closely related N. gonorrhoeae isolates, compared to Illumina sequencing. METHODS: N. gonorrhoeae strains collected from a London sexual health clinic were cultured and sequenced with MiSeq and MinION sequencing platforms. Accuracy was determined by comparing variant calls at 68 nucleotide positions (37 resistance-associated markers). Accuracy at varying MinION sequencing depths was determined through retrospective time-stamped read analysis. RESULTS: Of 22 MinION-MiSeq pairs reaching sufficient sequencing depth, agreement of variant call positions passing quality control criteria was 185/185 (100%; 95% confidence interval [CI], 98.0%-100.0%), 502/503 (99.8%; 95% CI, 98.9%-99.9%), and 564/565 (99.8%; 95% CI, 99.0%-100.0%) at 10x, 30x, and 40x MinION depth, respectively. Isolates identified as closely related by MiSeq, within one yearly evolutionary distance of ≤5 single nucleotide polymorphisms, were accurately identified via MinION. CONCLUSIONS: Nanopore sequencing shows utility as a rapid surveillance tool, identifying closely related N. gonorrhoeae strains, with just 10x sequencing depth, taking a median time of 29 minutes. This highlights its potential for tracking local transmission and AMR markers.

Anatomical appraisal of safe cholecystectomy: a prospective study.

BACKGROUND: Principles of safe cholecystectomy include dissection of the hepatocystic triangle and identification of the bilio-vascular structures to achieve critical view of safety. The aim of the present study was to document the variations in anatomical landmarks and bilio-vascular structures exposed during laparoscopic cholecystectomy. METHODS: All consecutive patients who underwent laparoscopic cholecystectomy were included in the study. Recommended techniques of safe cholecystectomy were followed. Rouviere's sulcus was classified into four types. Cystic duct was assessed for its length and diameter. A new classification was suggested for cystic artery. RESULTS: Five hundred patients were included in the study. Critical view of safety was achieved in 463 (92.6%) patients. Type1 Rouviere's sulcus was the most common variant found in 263 (52.6%). Normal cystic duct was present in 339 (67.8%). Wide cystic duct of normal length was the most common variant found in 71 (14.2%). Single cystic artery supero-medial to cystic duct (type1-s) was the most common variant found in 384 (76.8%). Inferior cystic artery i.e. artery infero-lateral to cystic duct (type-2) was the second most common variant, found in 40 (8%). Multiple cystic arteries in the hepatocystic triangle were due either to early division of single cystic artery (type-1m) or double cystic artery (type-3). Aberrant right hepatic artery giving off a small cystic artery (type-4) was seen in 12 (2.4%). CONCLUSION: Wide variation in the anatomical structures is noted when principles of safe laparoscopic cholecystectomy is followed. Awareness is paramount to avoid bilio-vascular injury during surgery.

Ogilvie's Syndrome with Perforation Peritonitis after Caesarean Section.

Acute colonic pseudo-obstruction in the immediate post-partum period, following Caesarean section is rare, and requires a high index of suspicion for diagnosis. Sometimes, rapidly progressive dilatation of the caecum can lead to perforation peritonitis. There are only a few case reports describing this important entity. Presented herein is a young primigravida, who developed Ogilvie's syndrome and peritonitis 5 days following an uneventful elective Caesarean section. Clinical details, management strategy adopted and a brief review of literature is presented to draw attention to this condition.

Laparoscopic surgery for right colon cancer with intestinal malrotation: A case report and review of the literature.

Intestinal malrotation (IM) results from abnormal midgut rotation during embryogenesis and usually presents in early infancy. IM in adults is often an incidental radiological diagnosis. Right colon cancer with IM has been sparingly reported in the world literature. Only 44.7% of these documented cases underwent successful laparoscopic resection, all of which were reported from Japan. The presence of aberrant anatomy and altered vascular orientation in IM presents challenges for laparoscopic resection with adequate nodal clearance. We present the case of a 72-year-old female diagnosed with carcinoma of the ascending colon, with IM incidentally detected on pre-operative cross-sectional imaging. She underwent laparoscopic right hemicolectomy. We also reviewed the documented cases of successful laparoscopic surgery for right colon cancers with IM. Aided by accurate delineation of vascular anatomy on pre-operative cross-sectional imaging, laparoscopic colectomy in the setting of IM is safe and feasible, and should be favourably considered.

Nerve stimulation guided bilateral pudendal nerve block versus landmark-based caudal block for hypospadias repair in young children: a prospective, randomized, pragmatic trial.

INTRODUCTION: Caudal block is frequently performed to provide analgesia for hypospadias repair. Literature suggests that pudendal block provides prolonged postoperative analgesia as compared with caudal block in children between 2 and 5 years. We compared the efficacy of pudendal and caudal blocks in children less than 2 years. METHODS: 60 children scheduled for hypospadias repair received standard general anesthesia along with either pudendal or caudal block (groups of 30 each). Variables collected were demographic data, block time, operating room time, intraoperative pain medication need, pain assessment score and medication need in the recovery room and pain assessment at home. RESULT: Groups were demographically similar. No differences were observed in the following recorded times (minutes): block procedure (caudal: 9.5±4.0, pudendal: 10.6±4.1, p=0.30), anesthesia (caudal: 17.3±5.3, pudendal: 17.7±4.3, p=0.75), total OR (caudal: 171±35, pudendal: 172±41; p=0.95) and postanesthesia care unit (PACU) stay (caudal: 88±37, pudendal: 86±42; p=0.80). Additionally, no differences were observed in rescue pain medication need in the operating room (caudal: 0, pudendal: 2 (p=0.49), in PACU (caudal: 4, pudendal: 4, p=0.99), pain assessed at home, time to pain level 2 (caudal: 13.93±8.9, pudendal: 15.17±8.7), average pain scores (p=0.67) and total pain free epochs (pain level of zero) (p=0.80) in the first 24 hours. DISCUSSION: In children less than 2 years, both blocks provide comparable intraoperative and postoperative pain relief in the first 24 hours after hypospadias surgery. TRIAL REGISTRATION NUMBER: NCT03145415.

Facilitators and barriers for clinical implementation of a 30-minute point-of-care test for Neisseria gonorrhoeae and Chlamydia trachomatis into clinical care: A qualitative study within sexual health services in England.

Point-of-care tests (POCTs) to diagnose sexually transmitted infections (STIs) have potential to positively impact patient management and patient perceptions of clinical services. Yet there remains a disconnect between development of new technologies and their implementation into clinical care. With the advent of new STI POCTs arriving to the global market, guidance for their successful adoption and implementation into clinical services is urgently needed. We conducted qualitative in-depth interviews with professionals prior to and post-implementation of a Chlamydia trachomatis/Neisseria gonorrhoeae POCT into clinical services in England to define key stakeholder roles and explore the process of POCT integration. Participants self-identified themselves as key stakeholders in the STI POCT adoption and/or implementation processes. Data consisted of interview transcripts, which were analysed thematically using NVIVO 11. Six sexual health services were included in the study; three of which have implemented POCTs. We conducted 40 total interviews: 31 prior to POCT implementation and 9 follow-up post-implementation. Post-implementation data showed that implementation plans required little or no change during service evaluation. Lead clinicians and managers self-identified as key stakeholders for the decision to purchase, while nurses self-identified as "change champions" for implementation. Many identified senior clinical staff as those most likely to introduce and drive change. However, participants stressed the importance of engaging all clinical staff in implementation. While the accuracy of the POCT, its positive impact on patient management and the ease of its integration within existing pathways were considered essential, costs of purchasing and utilising the technology were identified as central to the decision to purchase. Our study shows that key decision-makers for adoption and implementation require STI POCTs to have laboratory-comparable accuracy and be affordable for purchase and ongoing use. Further, successful integration of POCTs into sexual health services relies on supportive interpersonal relationships between all levels of staff.

Vaginal microbiota in ethnically diverse young women who did or did not develop pelvic inflammatory disease: community-based prospective study.

OBJECTIVES: A lactobacilli-dominated vaginal microbiome may protect against pelvic inflammatory disease (PID), but one dominated by Gardnerella species might increase susceptibility. Not all lactobacilli are equally protective. Recent research suggests that D(-) isomer lactic acid producing lactobacilli (Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus gasseri) may protect against infection with Chlamydia trachomatis, an important cause of PID. Lactobacillus iners , which produces L(+) isomer lactic acid, may be less protective. We investigated the microbiome in stored vaginal samples from participants who did or did not develop PID during the prevention of pelvic infection (POPI) chlamydia screening trial. METHODS: Long-read 16S rRNA gene nanopore sequencing was used on baseline vaginal samples (one per participant) from all 37 women who subsequently developed clinically diagnosed PID during 12-month follow-up, and 111 frequency matched controls who did not, matched on four possible risk factors for PID: age <20 versus ≥20, black ethnicity versus other ethnicity, chlamydia positive versus negative at baseline and ≥2 sexual partners in the previous year versus 0-1 partners. RESULTS: Samples from 106 women (median age 19 years, 40% black ethnicity, 22% chlamydia positive, 54% reporting multiple partners) were suitable for analysis. Three main taxonomic clusters were identified dominated by L. iners, L. crispatus and Gardnerella vaginalis. There was no association between a more diverse, G. vaginalis dominated microbiome and subsequent PID, although increased Shannon diversity was associated with black ethnicity (p=0.002) and bacterial vaginosis (diagnosed by Gram stain p<0.0001). Women who developed PID had similar relative abundance of protective D(-) isomer lactic acid producing lactobacilli to women without PID, but numbers of PID cases were small. CONCLUSIONS: In the first-ever community-based prospective study of PID, there was no clear association between the vaginal microbiome and subsequent development of PID. Future studies using serial samples may identify vaginal microbial communities that may predispose to PID.

Exploring beliefs and distress in patients with facial palsies.

Evidence suggests that people with facial palsy may experience higher levels of distress, but the reasons for this are yet to be explored. This study aimed to explore people's illness beliefs, emotions, and behaviours in relation to their facial palsy and understand how distress is experienced by this group. Semi-structured individual interviews were conducted in the UK with adults with facial palsy. Interview questions were theoretically informed by the Common-Sense Self-Regulatory Model (CS-SRM). Thematic Analysis was conducted following a combined inductive and deductive approach. Twenty people with facial palsy participated (70% female; aged 29-84). Patient distress was accounted for by illness beliefs (symptoms, cause, control and treatment, timeline and consequences), and four additional themes (coping behaviours, social support, identity and health service provision). Experiences of anxiety, depression, and anger were widespread, and some participants experienced suicidal ideation. The burden of managing a long-term condition, altered self-perception, and social anxiety and isolation were key drivers of distress. There is a need for more integrated psychological support for patients with facial palsy. Within clinical consultations, patient's beliefs about facial palsy should be identified and systematically addressed. Service development should include appropriate referral to specialist psychological support via an established care pathway.

Multiscale Approach for Computing Gated Ligand Binding from Molecular Dynamics and Brownian Dynamics Simulations.

We develop an approach to characterize the effects of gating by a multiconformation protein consisting of macrostate conformations that are either accessible or inaccessible to ligand binding. We first construct a Markov state model of the apo-protein from atomistic molecular dynamics simulations from which we identify macrostates and their conformations, compute their relative macrostate populations and interchange kinetics, and structurally characterize them in terms of ligand accessibility. We insert the calculated first-order rate constants for conformational transitions into a multistate gating theory from which we derive a gating factor γ that quantifies the degree of conformational gating. Applied to HIV-1 protease, our approach yields a kinetic network of three accessible (semi-open, open, and wide-open) and two inaccessible (closed and a newly identified, "parted") macrostate conformations. The parted conformation sterically partitions the active site, suggesting a possible role in product release. We find that the binding kinetics of drugs and drug-like inhibitors to HIV-1 protease falls in the slow gating regime. However, because γ = 0.75, conformational gating only modestly slows ligand binding. Brownian dynamics simulations of the diffusional association of eight inhibitors to the protease─having a wide range of experimental association constants (∼104-1010 M-1 s-1)─yields gated rate constants in the range of ∼0.5-5.7 × 108 M-1 s-1. This indicates that, whereas the association rate of some inhibitors could be described by the model, for many inhibitors either subsequent conformational transitions or alternate binding mechanisms may be rate-limiting. For systems known to be modulated by conformational gating, the approach could be scaled computationally efficiently to screen association kinetics for a large number of ligands.

The HIV-1 Nucleocapsid Regulates Its Own Condensation by Phase-Separated Activity-Enhancing Sequestration of the Viral Protease during Maturation.

A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular 'sponges', stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of ∼2400 Gag and ∼120 GagPol by the viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak-strong-moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules.

Median Arcuate Ligament Syndrome: an Important Consideration in Pancreaticoduodenectomy.

Pancreaticoduodenectomy (PD) is a common surgery performed with curative intent for periampullary and pancreatic head neoplasms. In the presence of intrinsic celiac artery narrowing due to atherosclerosis or extrinsic compression due to median arcuate ligament syndrome (MALS), division of the gastroduodenal artery during PD can result in liver ischemia. This report describes a patient who had MALS which was treated by intraoperative median arcuate ligament release during PD, resulting in restoration of hepatic artery pulsations. Preventive, management strategies for MALS and the communicating vascular arcades between the celiac and superior mesenteric arterial systems are discussed in the context of PD.

Twelve-Year Survival After Resection of Hepatocellular Carcinoma with Right Atrial Tumor Thrombus.

INTRODUCTION: A large hepatocellular carcinoma (HCC) with macrovascular invasion is generally considered to have poor prognosis due to unfavorable tumor biology andsuch patients are relegated to palliative options. This report describes long term survival after surgery in a patient with HCC and tumor thrombus in the rightatrium. METHODS: Case records of the patient, details of follow up visits and surveillance computed tomography scans performed were reviewed. A brief discussion of thesurgical strategy adopted along with outcome of similar cases in literature is presented. RESULTS: A 60 year old man presented with a HCC in segments 4, 5 with extension of tumor into the middle, left hepatic veins, inferior vena cava and right atrium.Patient underwent extraction of the tumor thrombus from the right atrium under cardiopulmonary bypass along with extended left hepatectomy. He receivedthree cycles of adjuvant chemotherapy with Gemcitabine, Oxaliplatin and Interferon. There was a parietal wall recurrence after 1 year and 10 months whichwas excised. Since then he remains well and is on regular follow up for more than 12 years from his index surgery with no evidence of disease, making himthe longest known survivor with such an advanced presentation. CONCLUSION: This is an unusual instance where aggressive resection for HCC with right atrial tumor thrombus has resulted in an exceptionally long survival.

Sexually transmitted infections and factors associated with risky sexual practices among female sex workers: A cross sectional study in a large Andean city.

BACKGROUND: There are limited published data on factors related to risky sexual practices (RSP) affecting sexually transmitted infections (STIs) among female sex workers (FSWs) in Ecuador. METHODS: Cross-sectional study of FSWs presenting for a consultation in a primary health care centre during 2017. A questionnaire was administered to collect information on RSP and potential risk factors including age, membership of an FSW association, self-report of previous STI diagnosis, previous treatment for suspected STI and temporary migration for sex work. Associations between RSP and potential risk factors were estimated by logistic regression. The proportion of STI was estimated from vaginal swabs by real-time PCR for four sexually transmitted pathogens (Neisseria gonorrhoeae, Trichomonas vaginalis, Chlamydia trachomatis, and Mycoplasma genitalium). RESULTS: Of 249 FSWs recruited, 22.5% had reported RSPs at least once during sex work. Among FSWs reporting unprotected vaginal sex in the previous three months, 25.5% had at least one other RSP type. 17.6% (95%CI 13.3-22.8) had at least one active STI. Prevalence of co-infections was 2.4% (95%CI 1.1-5.2). In multivariable analysis, RSP was associated with age (adjusted OR 1.06; 95%CI 1.02-1.10), membership of an FSWs association (aOR 3.51; 95%CI 1.60-7.72) and self-reported previous STI (aOR 3.43; 95%CI 1.28-9.17). CONCLUSIONS: Among a population of female sex workers with high proportion of STIs, increasing age and belonging to an FSWs association was associated with a higher likelihood of engaging in RSP with clients. Engaging with FSWs organisations may reduce the burden of STI among sex workers.

Conformational Analysis of Heparin-Analogue Pentasaccharides by Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulations.

Elucidation and improvement of the blood coagulant properties of heparin are the focus of intense research. In this study, we performed conformational analysis using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations on the heparin pentasaccharide analogue idraparinux, its disulfonatomethyl analogue, which features a slightly improved blood coagulation property, and a trisulfonatomethyl analogue, in which the activity has been totally abolished. As the ring conformation of the G subunit has been suggested as a major determinant of the biological properties, we analyzed the sugar ring conformations and dynamics of the interglycosidic linkages. We found that the conformation of the G ring is dominated by the 2SO skewed boat next to the 1C4 chair in all three derivatives. Both the thermodynamics and the kinetics of the conformational states were found to be highly similar in the three derivatives. Molecular kinetic analysis showed that the 2SO skewed boat state of the G ring is equally favorable in the three analogues, resulting in similar 2SO populations. Also, the transition kinetics from the 1C4 chair to the 2SO skewed boat was found to be comparable in the derivatives, which indicates a similar energy barrier between the two states of the G subunit. We also identified a slower conformational transition between the dominant 4C1 chair and the boat conformations on the E subunit. Both G and E ring flips are also accompanied by changes along the interglycosidic linkages, which take place highly synchronously with the ring flips. These findings indicate that conformational plasticity of the G ring and the dominance of the 2SO skewed boat populations do not necessarily warrant the biological activity of the derivatives and hence the impact of other factors also needs to be considered.

Novel composite trachea grafts using 3-dimensional printing.

Objective: Porcine-derived small intestine submucosa (SIS) extracellular matrix (ECM) surgical patches claim to have greater regenerative properties compared with dermal extracellular matrices. We hypothesized that using SIS-ECM in a bioengineered composite tracheal graft would allow better incorporation into the native tissue. Methods: Two types of size-matched polycaprolactone support scaffolds were designed: rigid and flexible. The SIS-ECM was wrapped around the polycaprolactone supports lining the inside and outside of the graft. The grafts were implanted in 4 Yorkshire pigs, replacing an ∼2 cm segment of native trachea. Airway patency was evaluated with computed tomography scans and explanted grafts were examined grossly and histologically. Results: All animals survived through the immediate postoperative period. Generally, extraluminal examination showed a smooth transition between native and graft without significant volumetric loss. Animals that received the flexible design survived ∼10 days longer than those that received the rigid design; however, severe perianastomotic intraluminal granulation tissue was observed. The rigid design had less significant intraluminal granulation tissue development at the distal anastomosis, but partial dehiscence had occurred at the proximal anastomosis interrupting graft incorporation. Conclusions: The generally good extraluminal graft incorporation in our composite tracheal graft highlights some increased regenerative capabilities of SIS-ECM. However, the presence of intraluminal granulation tissue indicates that its use as an off-the-shelf, unaltered substrate in an airway graft is still not ideal. Further research must be conducted to determine whether a modification of the substrate is possible to enhance luminal airway incorporation and to exert control over the mechanisms responsible for granulation tissue development.

High prevalence of coinfection of azithromycin-resistant Mycoplasma genitalium with other STIs: a prospective observational study of London-based symptomatic and STI-contact clinic attendees.

OBJECTIVES: Azithromycin treatment of Chlamydia trachomatis (CT) may not be adequate to treat concomitant Mycoplasma genitalium (MG) infection, and particularly if MG has macrolide resistance-associated mutations (MG-MRAMs). We estimated prevalence of coinfections of CT with MG carrying MRAM, and risk factors for MG-MRAM among a sexual health clinic population. STUDY DESIGN AND SETTING: Among symptomatic and STI-contact clinic attendees in London, prevalence of CT-MG coinfection and MG-MRAM were estimated using nucleic acid amplification testing and Sanger sequencing, respectively, and their associated risk factors analysed using logistic regression. RESULTS: MG prevalence was 7.5% (23/307), 17.3% (30/173), and 11.4% (8/70) in females, men who have sex with women (MSW) and men who have sex with men (MSM), respectively; MG coinfection in CT-infected participants represented 28.0% (7/25), 13.5% (5/37), 0.0% (0/0), respectively. Presence of MG-MRAM was 39.1% (9/23) in female swabs, 70.0% (21/30) in MSW urine and 83.3% (5/6) in MSM rectal swabs. In multivariate analyses, coinfection with another STI was strongly associated with MG-MRAM (OR: 7.19; 95% CI: 2.4 to 21.5). CONCLUSION: A significant proportion of participants in our study of symptomatic patients and STI contacts were infected with macrolide-resistant MG, suggesting that testing for MG and MRAM, for MG positives, might be clinically useful. The findings also suggest services explore potential benefits of testing CT positive samples for MG in these patient groups. Where MG testing is not available, potential high rates of MG coinfection should be borne in mind when considering azithromycin in the treatment of CT among STI contacts and symptomatic patients.

Successes and Failures in Tracheal Bioengineering: Lessons Learned.

BACKGROUND: Controversy in tracheal reconstruction using grafts and bioengineered constructs highlights the importance of animal studies before human application. Small animal models help to refine designs but do not adequately model sizes relevant to human anatomy. We have conducted extensive large animal studies and summarize our findings in 26 consecutive transplants. METHODS: We pooled 26 large animal studies together to investigate common elements related to successes and failures. In general the engineered tracheal graft consisted of a decellularized extracellular matrix surgical patch supported by a 3-dimensional-printed plastic polymer scaffold. Circumferential graft coverage ranged from 50% to 100%, spanning the length of 4 to 6 tracheal rings. Some grafts included embedded stem cells. Control grafts were fabricated without the support scaffold. At death grafts were harvested and examined grossly and through histology. RESULTS: The support scaffold prevented graft malacia and collapse. Luminal epithelialization was most extensive in grafts with smaller circumferential coverage. Smaller circumferential coverage was also associated with longest animal survival. Chondrogenesis was only observed in grafts with embedded stem cells. Survival time was shortest in 100% circumferential grafts. Granulation tissue was an issue for all graft designs. CONCLUSIONS: Large animal models capture challenges and complexities relevant to human anatomy. Development of granulation tissue remains a challenge, especially in circumferential grafts. Significant additional research is needed to investigate granulation tissue formation and to provide actionable insight into its management.