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Patients with congenital diaphragmatic hernia (CDH) can present with other congenital anomalies, but an associated choledochal cyst (CC) has rarely been described. The simultaneous diagnosis of both anomalies complicates patient management. There is no consensus on the ideal timing for CC excision. Unrepaired CC is associated with risks of developing biliary sludge, choledocholithiasis, and cholangitis. After a CDH repair with mesh, secondary bacterial translocation caused by a delayed CC repair could lead to mesh superinfection. Conversely, early CC surgical management could cause mesh displacement and lead to CDH recurrence, requiring reintervention. We present the rare case of a CC occurring in a neonate with a prenatally diagnosed right CDH. One month after an uncomplicated CDH repair with mesh, while the patient was still hospitalized for pulmonary hypertension, she developed progressive cholestasis and acholic stools. Investigations revealed a nonpreviously suspected CC. Conservative treatment was attempted, but CC perforation with secondary biliary peritonitis occurred. Open CC excision with a Roux-en-Y hepaticojejunostomy was therefore performed on day of life (DOL) 41. Having suffered no short-term surgical complications, the patient was discharged on DOL 83 because of prolonged ventilatory support due to pulmonary hypertension. Now 12 months after surgery, she is doing well with normal liver function tests and imaging studies. In summary, CC should be considered in the differential diagnosis of progressive cholestasis in patients with CDH. Surgical repair of a symptomatic CC should not be delayed even in the presence of mesh given the risks of CC complications., Competing Interests: Conflict of Interest None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. ( https://creativecommons.org/licenses/by/4.0/ ).)

Background: HBeAg loss is an important endpoint for antiviral therapy in chronic hepatitis B (CHB), however there are no reliable biomarkers to identify patients who will respond to the addition of pegylated interferon to nucleos(t)ide analogue (NA) therapy., Aim: To evaluate the use of serum biomarkers to predict HBeAg loss., Methods: HBeAg positive CHB participants on NAs who switched-to or added-on 48 weeks pegylated interferon alpha2b (clinicaltrial.gov NCT01928511) were evaluated at week 72 for HBeAg loss. The predictive ability of qHBeAg, qHBsAg, HBV RNA and clinical variables for HBeAg loss were investigated., Results: HBeAg loss occurred in 15/55 (27.3%) participants who completed 48 weeks of pegylated interferon. There was a lower baseline qHBeAg (1.18 IU/mL [2.27] versus 10.04 IU/mL [24.87], P = 0.007) among participants who lost HBeAg. Baseline qHBeAg (OR = 0.15, 95% CI 0.03-0.66, P = 0.01) and detectable HBV DNA at baseline (OR = 25.00, 95% CI 1.67-374.70, P = 0.02) were independent predictors of HBeAg loss. In addition, on-treatment qHBeAg was also a strong predictor of HBeAg loss (OR = 0.39, 95% CI 0.18-0.81, P = 0.012). The models combining detectable baseline HBV DNA with baseline (C-statistic 0.82) and on-treatment (C-statistic 0.83) had good accuracy for predicting HBeAg loss. A rise in qHBeAg ≥ 10 IU/ml was a predictor of flare (ALT ≥ 120 U/ml) on univariable analysis but not after adjustment for treatment arm., Conclusions: Baseline and on-treatment qHBeAg is a useful biomarker that can identify participants on NA therapy who may benefit from adding or switching to pegylated interferon., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.Daniel Q. Huang Advisory board: Eisai, Gilead. Liang Shen nil; Wah Phyo nil; Gavin Cloherty Employee of Abbott Diagnostics; Emily K. Butler Employee of Abbott Diagnostics; Mary C. Kuhns Employee of Abbott Diagnostics; Anne L. McNamara Employee of Abbott Diagnostics; Vera Holzmayer Employee of Abbott Diagnostics; Jeffrey Gersch Employee of Abbott Diagnostics; Mark Anderson Employee of Abbott Diagnostics; Wei Lyn Yang: Advisory board: Gilead, Abbie, Bristol Myers Squibb, MSD; Jing Hieng Ngu Advisory Board: Gilead Sciences; Speakers Bureau: Abbvie; Jason Chang nil; Jessica Tan nil; Taufique Ahmed nil; Yock Young Dan Advisory Board: BMS, Gilead, Novartis, Abbvie, MSD; Speaker's Bureau: Sanofi Aventis, Siemens; Research grants: BMS, Novartis and Johnson & Johnson; Yin Mei Lee nil; Guan Huei Lee nil; Poh Seng Tan nil; Htet Toe Wai Khine nil; Amy Tay nil; Seng Gee Lim: Advisory Board: Gilead Sciences, Roche, GSK, Janssen, Grifols, Assembly, Arbutus, Abbott, Sysmex. Speakers Bureau: Gilead Sciences, Abbott, Sysmex, Roche, GSK. Educational/research funding: Abbott, Gilead Sciences., (Copyright © 2024. Published by Elsevier B.V.)

Background: Tracheostomy placement is much more common in adults than children following severe trauma. We evaluated whether tracheostomy rates and outcomes differ for pediatric patients treated at trauma centers that primarily care for children versus adults., Methods: We conducted a retrospective cohort study of patients younger than 18 years in the National Trauma Data Bank from 2007 to 2016 treated at a Level I/II pediatric, adult, or combined adult/pediatric trauma center, ventilated >24 hours, and who survived to discharge. We used multivariable logistic regression adjusted for age, insurance, injury mechanism and body region, and Injury Severity Score to estimate the association between the three trauma center types and tracheostomy. We used augmented inverse probability weighting to model the likelihood of tracheostomy based on the propensity for treatment at a pediatric, adult, or combined trauma center, and estimated associations between trauma center type with length of stay and postdischarge care., Results: Among 33,602 children, tracheostomies were performed in 4.2% of children in pediatric centers, 7.8% in combined centers (adjusted odds ratio [aOR], 1.47; 95% confidence interval [CI], 1.20-1.81), and 11.2% in adult centers (aOR, 1.81; 95% CI, 1.48-2.22). After propensity matching, the estimated average tracheostomy rate would be 62.9% higher (95% CI, 37.7-88.1%) at combined centers and 85.3% higher (56.6-113.9%) at adult centers relative to pediatric centers. Tracheostomy patients had longer hospital stay in pediatric centers than combined (-4.4 days, -7.4 to -1.3 days) or adult (-4.0 days, -7.2 to -0.9 days) centers, but fewer children required postdischarge inpatient care (70.1% pediatric vs. 81.3% combined [aOR, 2.11; 95% CI, 1.03-4.31] and 82.4% adult centers [aOR, 2.51; 95% CI, 1.31-4.83])., Conclusion: Children treated at pediatric trauma centers have lower likelihood of tracheostomy than children treated at combined adult/pediatric or adult centers independent of patient or injury characteristics. Better understanding of optimal indications for tracheostomy is necessary to improve processes of care for children treated throughout the pediatric trauma system., Level of Evidence: Prognostic and Epidemiological; Level III., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

The development and application of modern sequencing technologies have led to many new improvements in food safety and public health. With unprecedented resolution and big data, high-throughput sequencing (HTS) has enabled food safety specialists to sequence marker genes, whole genomes, and transcriptomes of microorganisms almost in real-time. These data reveal not only the identity of a pathogen or an organism of interest in the food supply but its virulence potential and functional characteristics. HTS of amplicons, allow better characterization of the microbial communities associated with food and the environment. New and powerful bioinformatics tools, algorithms, and machine learning allow for development of new models to predict and tackle important events such as foodborne disease outbreaks. Despite its potential, the integration of HTS into current food safety systems is far from complete. Government agencies have embraced this new technology, and use it for disease diagnostics, food safety inspections, and outbreak investigations. However, adoption and application of HTS by the food industry have been comparatively slow, sporadic, and fragmented. Incorporation of HTS by food manufacturers in their food safety programs could reinforce the design and verification of effectiveness of control measures by providing greater insight into the characteristics, origin, relatedness, and evolution of microorganisms in our foods and environment. Here, we discuss this new technology, its power, and potential. A brief history of implementation by public health agencies is presented, as are the benefits and challenges for the food industry, and its future in the context of food safety., (© 2022. The Author(s).)

In most low- and middle-income countries, trauma registries are uncommon. Although institutional registries for all trauma patients are ideal, it can be more practical to institute departmental registries for specific subsets of patients. Komfo Anokye Teaching Hospital (KATH) has started a locally developed, self-funded orthopaedic trauma registry. We describe methods and experiences for data collection and examine patient and injury characteristics, data quality, and the utility of the registry. Of 961 individuals in the registry, 67.9% were males, and the median age was 40 years. Motor vehicle collision (23.3%) was the most frequent mechanism of injury. Lower extremity fractures were the most common injury (60.6%), and 43.9% of injuries were managed operatively. Data quality was reasonable with missingness under 10% for 13 of 14 key variables, with inconsistencies of dates of injury, admission, treatment, and discharge in 9.1% of cases. However, the type of operation was missing for 73.2% of operative cases. Despite these limitations, the registry has been used for quality improvement and to successfully advocate for resources to improve trauma care. The registry has been improved by adding more detailed outcome variables, creating a standardised codebook of categorical variables, and adding more fields to allow for multiple injuries. In conclusion, it is practical and sustainable to institute a locally developed, self-funded orthopaedic trauma registry in Ghana that provides data with reasonable quality. Such a registry can be used to advocate for more resources to care for injured patients adequately and for quality improvement., Funding: None declared., Competing Interests: Conflict of interest: None declared, (Copyright © The Author(s).)

Objective: The objective of this study was to examine an academic air ambulance service's experience with prehospital transfusion of plasma and red blood cells in pediatric trauma for evidence of efficacy on the treatment of shock and coagulopathy., Methods: All trauma patients < 18 years old transfused during transport by the University of Washington Airlift Northwest (Airlift) air medical transport service to Harborview Medical Center, Seattle, WA, were identified. Controls were matched 1:1 from pediatric trauma patients transported by Airlift before transfusion support became available. Demographics, injury scores, emergency department admission and interval laboratory values, blood product use, and hospital outcome measures were registered., Results: Seventeen cases met the inclusion criteria and were matched by age and Injury Severity Score to 17 control patients (mean age = 10.5 vs. 10.9 years; New Injury Severity Score, 37 vs. 40.7). No significant differences in vital signs, shock index, or mortality were observed. Cases received less in-flight crystalloid (4.3 mL/kg vs. 16.9 mL/kg, P = .004), had higher admission fibrinogen levels (238 vs. 148mg/dL, P = .007), and shorter time to normalization of the international normalized ratio (6.4 vs. 19.1 hours, P = .04)., Conclusions: In this small series, hemostatic resuscitation during air medical transport was associated with less crystalloid administration and better support of coagulation indices., (Copyright © 2021 Air Medical Journal Associates. Published by Elsevier Inc. All rights reserved.)

Purpose: To determine the population-based rate of emergency surgery performed in Ghana, categorized by hospital level., Methods: Data on operations performed from June 2014 to May 2015 were obtained from a nationally representative sample of hospitals and scaled up to nationwide estimates. Operations were categorized as to: "emergency" or "elective" and as to "essential" (most cost-effective, highest population impact) or "other" according to the World Bank's Disease Control Priorities project., Results: Of 232,776 (95% UI 178,004-287,549) total operations performed nationally, 48% were emergencies. 112,036 emergency operations (95% UI 92,105-131,967) were performed and the annual national rate was 416 per 100,000 population (95% UI 342-489). Most emergency operations (87%) were in the essential category. Of essential emergency procedures, 47% were obstetric and gynecologic, 22% were general surgery, and 31% were trauma. District (first-level) hospitals performed 54%, regional hospitals 10%, and tertiary hospitals 36% of all emergency operations. About half (54%) of district hospitals did not have a fully trained surgeon, however, these hospitals performed 36% of district hospital emergency operations and 20% of all emergency operations., Conclusions: Emergency operations make up nearly half of all operations performed in Ghana. Most are performed at district hospitals, many of which do not have fully trained surgeons. Obstetric procedures make up a large portion of emergency operations, indicating a need for improved provision of non-obstetric emergency surgical care. These data are useful for future benchmarking efforts to improve availability of emergency surgical care in Ghana and other low- and middle-income countries., (© 2019. Springer-Verlag GmbH Germany, part of Springer Nature.)

Objectives: To evaluate the optimal timing of tracheostomy for injured adolescents., Design: Retrospective cohort study., Setting: Trauma facilities in the United States., Patients: Adolescents (age 12-17 yr) in the National Trauma Data Bank (2007-2016) who were ventilated for greater than 24 hours and survived to discharge., Interventions: None., Measurements and Main Results: After stratifying by traumatic brain injury diagnosis, we compared ICU and hospital length of stay, pneumonia, and discharge disposition of patients with tracheostomy prior to three cut points (3, 7, and 14 d after admission) to 1) patients intubated at least as long as each cut point and 2) patients with tracheostomy on or after each cut point. Of 11,045 patients, 1,391 (12.6%) underwent tracheostomy. Median time to tracheostomy was 9 days (interquartile range, 6-13 d) for traumatic brain injury and 7 days (interquartile range, 3-12 d) for nontraumatic brain injury patients. Nontraumatic brain injury patients with tracheostomy prior to 7 days had 5.6 fewer ICU days (-7.8 to -3.5 d) and 5.7 fewer hospital days (-8.8 to -2.7 d) than patients intubated greater than or equal to 7 days and had 14.8 fewer ICU days (-19.6 to -10.0 d) and 15.3 fewer hospital days (-21.7 to -8.9 d) than patients with tracheostomy greater than or equal to 7 days. Similar differences were observed at 14 days but not at 3 days for both traumatic brain injury and nontraumatic brain injury patients. At the 3- and 7-day cut points, both traumatic brain injury and nontraumatic brain injury patients with tracheostomy prior to the cut point had lower risk of pneumonia and risk of discharge to a facility than those with tracheostomy after the cut point., Conclusions: For injured adolescents, tracheostomy less than 7 days after admission was associated with improved in-hospital outcomes compared with those who remained intubated greater than or equal to 7 days and with those with tracheostomy greater than or equal to 7 days. Tracheostomy between 3 and 7 days may be the optimal time point when prolonged need for mechanical ventilation is anticipated; however, unmeasured consequences of tracheostomy such as long-term complications and care needs must also be considered., Competing Interests: Drs. Butler, Killien, Vavilala, and Rivara received support for article research from the National Institutes of Health (NIH). Drs. Killien’s and Rivara’s institutions received funding from the NIH. Drs. Butler and Killien were supported by the NIH T-32 Pediatric Injury Research Training Program (5T32HD057822-09). The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2021 by the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies.)

Firearm storage method is a potentially modifiable risk factor for suicide. Using data from a large, multi-state survey, we sought to determine whether there is an association between mental health and household firearm storage practices, and characterize that association by state of residence. Participants who endorsed the presence of a household firearm and answered the mental health questions in the 2016-2017 Behavioral Risk Factor Surveillance System from eight states were included (n=26,949). Exposures were recent poor mental health (≥14 vs. 0-13 days/past month), and diagnosis of depression. Outcomes were household firearm storage practices (loaded, and both loaded and unlocked). Using Poisson regression, we calculated adjusted prevalence ratios (aPR) overall and stratified by state of residence. Of adults endorsing a household firearm, 35.1% reported storing a firearm loaded, and of those, 53.4% reported that the firearm was both loaded and unlocked. Neither recent poor mental health nor depression was associated with loaded (aPR 1.14 [95% CI: 0.95-1.37] and aPR 0.94 [95% CI 0.80-1.09], respectively) or loaded and unlocked (aPR 1.08 [95% CI 0.88-1.42] and aPR 1.04 [95% CI 0.88-1.22], respectively) firearm storage. In the setting of highly prevalent loaded firearm storage, no differences in storage practices by mental health indicators were observed across eight states despite disparate firearm policies and local culture. The lack of difference in storage practices by mental health indicators across several states highlights an opportunity to improve means safety counseling practices, and the need for dedicated evaluation of state-level firearm storage policies., (Published by Elsevier Inc.)

Background: No guidelines exist for management of hemodynamically stable children with suspected hollow viscus injury. We sought to determine factors contributing to surgeon management of these patients., Methods: Surgeon members of the Eastern Association for the Surgery of Trauma and American Pediatric Surgical Association completed a survey on 3 blunt abdominal injury scenarios: (1) isolated, (2) with multisystem injury, and (3) with traumatic brain injury (TBI), and a penetrating injury scenario. Multivariable logistic regression was used to determine factors associated with initial management of observation vs. operation for blunt injury and observation vs. local wound exploration versus laparoscopy for penetrating injury., Results: Of 394 surgeons (response rate 22.3%), 50.3% were pediatric surgeons. For scenarios 1-3, 32.2%, 49.3%, and 60.7% of surgeons chose operation over observation, respectively. Compared to isolated blunt injury, surgeons were more likely to choose operation for patients with multisystem injury (aOR 2.20, 95%CI: 1.78-2.72) or TBI (aOR 3.60, 95%CI: 2.79-4.66). Pediatric surgeons were less likely to choose operation (aOR 0.32, 95%CI: 0.22-0.44). For penetrating injury, 39.1%, 29.5%, and 31.5% of surgeons chose observation, local wound exploration, and laparoscopy, respectively., Conclusions: Large variation exists in management of hemodynamically stable children with suspected hollow viscus injury. Although patient injury characteristics account for some variation, surgeon factors such as type of surgeon also play a role. Evidence-based practice guidelines should be developed to standardize care., Type of Study: Cross-Sectional Survey., Level of Evidence: N/A., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Biomarkers such as quantitative HBsAg (qHBsAg), quantitative hepatitis B virus (HBV) core-related antigen (qHBcrAg) and HBV RNA may be useful in predicting HBsAg loss in patients with chronic hepatitis B (CHB) undergoing antiviral therapy., Aim(s): Our study evaluated qHBsAg, HBV RNA and qHBcrAg as a posthoc analysis of a randomized clinical trial of peginterferon±NA to determine their utility in predicting HBsAg loss., Methods: CHB patients who completed therapy with 48weeks peginterferon alpha2b ± nucleoside analogue therapy (clinicaltrial.gov NCT01928511) were evaluated at week 72 for HBsAg loss. The predictive ability of qHBsAg, qHBcrAg, HBV RNA and other variables were investigated by univariate and multivariate logistic models for HBeAg-negative patients by odds ratios, area under the curve (AUC), sensitivity, specificity, and positive and negative likelihood ratios (LR)., Results: HBsAg loss occurred in 15/114(13%) HBeAg-negative CHB patients who completed 48 weeks of peginterferon. At baseline, qHBsAg was superior to HBcrAg and HBV RNA with AUC 0.916, 0.649 and 0.542, respectively. Using multivariate analysis, the model comprising treatmentarm, age, gender, baseline qHBsAg, HBcrAg and HBV RNA, weeks 4 & 8 qHBsAg had the highest AUC(0.98), but the univariate model with week 8 qHBsAg <70 IU/mL had AUC 0.96. Hence, the contributions of variables other than qHBsAg were marginal. HBV RNA and qHBcrAg were weak predictors of HBsAg loss. Kinetics of the novel markers showed only qHBsAg had a good relationship with HBsAg loss while HBV RNA had a marginal relationship and HBcrAg did not change at all, and none had a good relationship with viral rebound., Conclusions: On-treatment biomarker predictors were better than baseline ones, and the best predictor of HBsAg loss at 72 weeks was week 8 qHBsAg <70 IU/mL., (© 2020 John Wiley & Sons Ltd.)

Background: There is minimal evidence evaluating the risks and benefits of laparoscopy use in hemodynamically stable children with suspected abdominal injuries. The objective of this study was to evaluate postoperative outcomes in a large cohort of hemodynamically stable pediatric patients with blunt abdominal injury., Methods: Using the 2015-2016 National Trauma Data Bank, all patients aged <18 y with injury severity score (ISS) ≤25, Glasgow coma scale ≥13, and normal blood pressure who underwent an abdominal operation for blunt abdominal trauma were included. Patients were grouped into three treatment groups: laparotomy, laparoscopy, and laparoscopy converted to laparotomy. Treatment effect estimation with inverse probability weighting was used to determine the association between treatment group and outcomes of interest., Results: Of 720 patients, 504 underwent laparotomy, 132 underwent laparoscopy, and 84 underwent laparoscopy converted to laparotomy. The median age was 10 (IQR: 7-15) y, and the median ISS was 9 (IQR: 5-14). Mean hospital length of stay was 2.1 d shorter (95% confidence interval [CI]: 0.9-3.2 d) and mean intensive care unit length of stay was 1.1 d shorter (95% CI: 0.6-1.5 d) for the laparoscopy group compared with the laparotomy group. The laparoscopy group had a 2.0% lower mean probability of surgical site infection than the laparotomy group (95% CI: 1.0%-3.0%)., Conclusions: In this cohort of hemodynamically stable pediatric patients with blunt abdominal injury, laparoscopy may have improved outcomes over laparotomy., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bacterial cell wall synthesis is an essential process in bacteria and one of the best targets for antibiotics. A critical step on this pathway is the export of the lipid-linked cell wall monomer, Lipid II, by its transporter MurJ. The mechanism by which MurJ mediates the transbilayer movement of Lipid II is not understood because intermediate states of this process have not been observed. Here we demonstrate a method to capture and detect interactions between MurJ and its substrate Lipid II by photo-cross-linking and subsequent biotin-tagging. We show that this method can be used to covalently capture intermediate transport states of Lipid II on MurJ in living cells. Using this strategy we probed several lethal arginine mutants and found that they retain appreciable substrate-binding ability despite being defective in Lipid II transport. We propose that Lipid II binding to these residues during transport induces a conformational change in MurJ required to proceed through the Lipid II transport cycle. The methods described to detect intermediate transport states of MurJ will be useful for characterizing mechanisms of inhibitors.

Objectives: To determine if higher fresh frozen plasma and platelet to packed RBC ratios are associated with lower 24-hour mortality in bleeding pediatric trauma patients., Design: Retrospective cohort study using the Pediatric Trauma Quality Improvement Program Database from 2014 to 2016., Setting: Level I and II pediatric trauma centers participating in the Trauma Quality Improvement Program PATIENTS:: Injured children (≤ 14 yr old) who received massive transfusion (≥ 40 mL/kg total blood products in 24 hr). Of 123,836 patients, 590 underwent massive transfusion, of which 583 met inclusion criteria., Interventions: None., Measurements and Main Results: Ratios of fresh frozen plasma:packed RBC and platelet:packed RBC. Of the 583 patients, 60% were male and the median age was 5 years (interquartile range, 2-10 yr). Overall mortality was 19.7% (95% CI, 16.6-23.2%) at 24 hours. There was 51% (adjusted relative risk, 0.49; 95% CI, 0.27-0.87; p = 0.02) and 40% (adjusted relative risk, 0.60; 95% CI, 0.39-0.92; p = 0.02) lower risk of death at 24 hours for the high (≥ 1:1) and medium (≥ 1:2 and < 1:1) fresh frozen plasma:packed RBC ratio groups, respectively, compared with the low ratio group (< 1:2). Platelet:packed RBC ratio was not associated with mortality (adjusted relative risk, 0.94; 95% CI, 0.51-1.71; p = 0.83)., Conclusions: Higher fresh frozen plasma ratios were associated with lower 24-hour mortality in massively transfused pediatric trauma patients. The platelet ratio was not associated with mortality. Although these findings represent the largest study evaluating blood product ratios in pediatric trauma patients, prospective studies are necessary to determine the optimum blood product ratios to minimize mortality in this population.

Background: Abdominal operations account for a majority of surgical volume in low-income countries, yet population-level prevalence data on surgically treatable abdominal conditions are scarce., Objective: In this study, our objective was to quantify the burden of surgically treatable abdominal conditions in Uganda., Methods: In 2014, we administered a two-stage cluster-randomized Surgeons OverSeas Assessment of Surgical Need survey to 4,248 individuals in 105 randomly selected clusters (representing the national population of Uganda)., Findings: Of the 4,248 respondents, 185 reported at least one surgically treatable abdominal condition in their lifetime, giving an estimated lifetime prevalence of 3.7% (95% CI: 3.0 to 4.6%). Of those 185 respondents, 76 reported an untreated condition, giving an untreated prevalence of 1.7% (95% CI: 1.3 to 2.3%). Obstructed labor (52.9%) accounted for most of the 238 abdominal conditions reported and was untreated in only 5.6% of reported conditions. In contrast, 73.3% of reported abdominal masses were untreated., Conclusions: Individuals in Uganda with nonobstetric abdominal surgical conditions are disproportionately undertreated. Major health system investments in obstetric surgical capacity have been beneficial, but our data suggest that further investments should aim at matching overall surgical care capacity with surgical need, rather than focusing on a single operation for obstructed labor., Competing Interests: The authors have no competing interests to declare., (© 2019 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/.)

Background: Uganda's ageing population (age 50 years and older) will nearly double from 2015 to 2050. HIV/AIDS, diabetes, stroke among other disease processes have been studied in the elderly population. However, the burden of disease from surgically-treatable conditions is unknown., Objectives: To determine the proportion of adults above 50 years with unmet surgical need and deaths attributable to probable surgically-treatable conditions., Methods: A cluster randomized sample representing the national population of Uganda was enumerated. The previously validated Surgeons Overseas assessment of surgical need instrument, a head-to-toe verbal interview, was used to determine any surgically-treatable conditions in two randomly-selected living household members. Deaths were detailed by heads of households. Weighted metrics are calculated taking sampling design into consideration and Taylor series linearization was used for sampling error estimation., Results: The study enumerated 425 individuals above age 50 years. The prevalence proportion of unmet surgical need was 27.8% (95%CI, 22.1-34.3). This extrapolates to 694,722 (95%CI, 552,279-857,157) individuals living with one or more surgically treatable conditions. The North sub-region was observed to have the highest prevalence proportion. Nearly two out of five household deaths (37.9%) were attributed to probable surgically treatable causes., Conclusion: There is disproportionately high need for surgical care among the ageing population of Uganda with approximately 700,000 consultations needed.

Treatment of chronic hepatitis B (CHB) patients with nucleos(t)ide analogs (NAs) suppresses hepatitis B virus (HBV) DNA synthesis but does not affect synthesis of HBV pregenomic RNA (pgRNA). Hepatitis B virus pgRNA is detectable in the serum during NA treatment and has been proposed as a marker of HBV covalently closed circular DNA activity within the infected hepatocyte. We developed an automated assay for the quantification of serum HBV pgRNA using a dual-target real-time quantitative PCR approach on the Abbott m2000sp/rt system. We demonstrate accurate detection and quantification of serum HBV RNA. Hepatitis B virus DNA was quantified using the Abbott RealTime HBV viral load assay. We further compared serum nucleic acid levels and kinetics in HBV-positive populations. Samples included on-therapy CHB samples (n = 16), samples (n = 89) from 10 treatment naïve CHB subjects receiving 12 weeks of NA treatment with 8-week follow-up, hepatitis B surface antigen-positive blood donor samples (n = 102), and three seroconversion series from plasmapheresis donors (n = 79 samples). Conclusion: During NA treatment of CHB subjects, we observed low correlation of HBV DNA to pgRNA levels; pgRNA concentration was generally higher than HBV DNA concentrations. In contrast, when NA treatment was absent we observed serum pgRNA at concentrations that correlated to HBV DNA and were approximately 2 log lower than HBV DNA. Importantly, we observe this trend in untreated subject samples from both chronic infections and throughout seroconversion during acute infection. Results demonstrate that the presence of pgRNA in serum is part of the HBV lifecycle; constant relative detection of pgRNA and HBV DNA in the serum is suggestive of a linked mechanism for egress for HBV DNA or pgRNA containing virions., (© 2018 by the American Association for the Study of Liver Diseases.)

Background: In low and middle-income countries, approximately 85% of children have a surgically treatable condition before the age of 15. Within these countries, the burden of pediatric surgical conditions falls heaviest on those in rural areas. The objective of the current study was to evaluate the relationship between rurality, surgical condition and treatment status among a cohort of Ugandan children., Methods: We identified 2176 children from 2315 households throughout Uganda using the Surgeons OverSeas Assessment of Surgical Need (SOSAS) survey. Children were randomly selected and were included in the study if they were 18 years of age or younger and had a surgical condition. Location of residence, surgical condition, and treatment status was compared among children., Results: Of the 305 children identified with surgical conditions, 81.9% lived in rural areas. The most prevalent causes of surgical conditions reported among rural and urban children were masses (24.0% and 25.5%, respectively), followed by wounds due to injury (19.6% and 16.4%, respectively). Among children with untreated surgical conditions, 79.1% reside in rural areas while 20.9% reside in urban areas. Among children with untreated surgical conditions, the leading reason for not seeking surgical care among children living in both rural and urban areas was a lack of money (40.6% and 31.4%, respectively), and the leading reason for not receiving care in both rural and urban settings was a lack of money (48.0% and 42.8%, respectively)., Conclusions: Our data suggest that over half of the children with a surgical condition surveyed are not receiving surgical care and a large majority of children with surgical needs were living in rural areas. Future interventions aimed at increasing surgical access in rural areas in low-income countries are needed., Competing Interests: Although funding was partially provided by Johnson and Johnson, this does not alter our adherence to all of PLOS ONE policies on sharing data and materials.

Hepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), infects an estimated 15-20 million people worldwide and confers a greater risk for accelerated progression to liver disease. However, limited HDV surveillance data are available in sub-Saharan Africa where HDV diversity is high. To determine the prevalence and diversity of HDV in Cameroon, serological and molecular characterization was performed on 1928 HBsAg positive specimens selected from retrospective viral surveillance studies conducted in Cameroon from 2010-2016. Samples were screened for HDV antibodies on the Abbott ARCHITECT instrument and for HDV RNA on the Abbott m2000 instrument by research assays. HDV positive specimens with sufficient viral load were selected for genomic sequencing. The seroprevalence of HDV in HBsAg positive samples from Cameroon was 46.73% [95% CI; 44.51-48.96%], with prevalence of active HDV infection being 34.2% [95% CI; 32.09-36.41%]. HDV genotypes 1, 6, 7 and 8 were identified amongst N = 211 sequences, including N = 145 genomes. HDV prevalence is high within the study cohort, indicating that a large portion of HBV infected individuals in Cameroon are at elevated risk for severe hepatitis and death. Collectively, these results emphasize the need for HBV vaccination and HDV testing in HBsAg positive patients in Cameroon.

Background: Blood safety and transfusion-transmitted infections (TTIs) are a major concern in low-resource areas. Laboratory screening of donors, a key contributor to blood safety, is usually done by enzyme-linked immunosorbent assay (ELISA) methods, which use expensive reagents and necessitate complex instruments and sophisticated laboratory staff. Rapid diagnostic tests (RDTs) are less expensive and easier to perform but have less sensitivity. Pathogen reduction technology (PRT) reduces transfusion transmission of malaria and may be effective in decreasing other TTIs. We explored the potential to improve blood safety by combining PRT and RDTs in comparison with current ELISA testing., Study Design and Methods: We identified the sensitivity of RDTs available in Uganda and the sensitivity of currently used ELISA. Data from a riboflavin-and-UV-based photochemical treatment PRT were used. Human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and malaria were studied. Probability models were developed for estimation of the number of infectious units of blood for each of these four infections using either current ELISA or the combination of RDT and PRT., Results: Compared to currently used ELISA, the combination of RDTs and PRT could reduce the rate of infectious units by 100, 20, 98, and 83% for HIV, HBV, HCV, and malaria, respectively, and would prevent use of 758 units of infectious blood per 10,000 units transfused., Conclusion: The combination of RDTs and PRT may improve blood safety in low-resource areas., (© 2018 AABB.)

Worldwide, an estimated 5% of hepatitis B virus (HBV) infected people are coinfected with hepatitis delta virus (HDV). HDV infection leads to increased mortality over HBV mono-infection, yet HDV diagnostics are not widely available. Prototype molecular (RNA) and serologic (IgG) assays were developed for high-throughput testing on the Abbott m2000 and ARCHITECT systems, respectively. RNA detection was achieved through amplification of a ribozyme region target, with a limit of detection of 5 IU/ml. The prototype serology assay (IgG) was developed using peptides derived from HDV large antigen (HDAg), and linear epitopes were further identified by peptide scan. Specificity of an HBV negative population was 100% for both assays. A panel of 145 HBsAg positive samples from Cameroon with unknown HDV status was tested using both assays: 16 (11.0%) had detectable HDV RNA, and 23 (15.7%) were sero-positive including the 16 HDV RNA positive samples. Additionally, an archival serial bleed panel from an HDV superinfected chimpanzee was tested with both prototypes; data was consistent with historic testing data using a commercial total anti-Delta test. Overall, the two prototype assays provide sensitive and specific methods for HDV detection using high throughput automated platforms, allowing opportunity for improved diagnosis of HDV infected patients.

Background: The Surgeons OverSeas Assessment of Surgical Need tool (SOSAS) was created to evaluate the burden of surgically treatable conditions in low- and middle-income countries. The goal of our study is to describe the face, head, and neck (FHN) conditions that need surgical care in Uganda, along with barriers to that care and disability from these conditions., Methods: A 2-stage cluster randomized SOSAS survey was administered in a cross-sectional manner between August and September 2014. Participants included randomly selected persons in 105 enumeration areas in 74 districts throughout Uganda with 24 households in each cluster. The SOSAS survey collected demographic and clinical data on all respondents. Univariate and multivariate logistic models evaluated associations of demographic characteristics and clinical characteristics of the FHN conditions and outcomes of whether health care was sought or surgical care was received., Results: Of the 4428 respondents, 331 (7.8%) reported having FHN conditions. The most common types of conditions were injury-related wounds. Of those who reported an FHN condition, 36% reported receiving no surgical care whereas 82.5% reported seeking health care. In the multivariate model, literacy and type of condition were significant predictors of seeking health care whereas village type, literacy, and type of condition remained significant predictors of receiving surgical care., Conclusions: Many individuals in Uganda are not receiving surgical care and barriers include costs, rural residency, and literacy. Our study highlights the need for targeted interventions in various parts of Uganda to increase human resources for surgery and expand surgical capacity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: In low- and middle-income countries (LMICs), an estimated 85% of children do not have access to surgical care. The objective of the current study was to determine the geographic distribution of surgical conditions among children throughout Uganda., Methods: Using the Surgeons OverSeas Assessment of Surgical Need (SOSAS) survey, we enumerated 2176 children in 2315 households throughout Uganda. At the district level, we determined the spatial autocorrelation of surgical need with geographic access to surgical centers variable., Findings: The highest average distance to a surgical center was found in the northern region at 14.97km (95% CI: 11.29km-16.89km). Younger children less than five years old had a higher prevalence of unmet surgical need in all four regions than their older counterparts. The spatial regression model showed that distance to surgical center and care availability were the main spatial predictors of unmet surgical need., Interpretation: We found differences in unmet surgical need by region and age group of the children, which could serve as priority areas for focused interventions to alleviate the burden. Future studies could be conducted in the northern regions to develop targeted interventions aimed at increasing pediatric surgical care in the areas of most need., Level of Evidence: Level III., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Multilevel posterior spine fusion is associated with significant intraoperative blood loss. Tranexamic acid is an antifibrinolytic agent that reduces intraoperative blood loss. The goal of this study was to compare the percent of total blood volume lost during posterior spinal fusion (PSF) with or without tranexamic acid in patients with adolescent idiopathic scoliosis (AIS)., Methods: Thirty-six AIS patients underwent PSF in 2011-2014; the last half (n=18) received intraoperative tranexamic acid. We retrieved relevant demographic, hematologic, intraoperative and outcomes information from medical records. The primary outcome was the percent of total blood volume lost, calculated from estimates of intraoperative blood loss (numerator) and estimated total blood volume per patient (denominator, via Nadler's equations). Unadjusted outcomes were compared using standard statistical tests., Results: Tranexamic acid and no-tranexamic acid groups were similar (all p>0.05) in mean age (16.1 vs. 15.2 years), sex (89% vs. 83% female), body mass index (22.2 vs. 20.2 kg/m2), preoperative hemoglobin (13.9 vs. 13.9 g/dl), mean spinal levels fused (10.5 vs. 9.6), osteotomies (1.6 vs. 0.9) and operative duration (6.1 hours, both). The percent of total blood volume lost (TBVL) was significantly lower in the tranexamic acid-treated vs. no-tranexamic acid group (median 8.23% vs. 14.30%, p = 0.032); percent TBVL per level fused was significantly lower with tranexamic acid than without it (1.1% vs. 1.8%, p=0.048). Estimated blood loss (milliliters) was similar across groups., Conclusions: Tranexamic acid significantly reduced the percentage of total blood volume lost versus no tranexamic acid in AIS patients who underwent PSF using a standardized blood loss measure.Level of Evidence: 3. Institutional Review Board status: This medical record chart review (minimal risk) study was approved by the University of Minnesota Institutional Review Board.

Objective: To quantify the burden of surgical conditions in Uganda., Background: Data on the burden of disease have long served as a cornerstone to health policymaking, planning, and resource allocation. Population-based data are the gold standard, but no data on surgical burden at a national scale exist; therefore, we adapted the Surgeons OverSeas Assessment of Surgical Need survey and conducted a nation-wide, cross-sectional survey of Uganda to quantify the burden of surgically treatable conditions., Methods: The 2-stage cluster sample included 105 enumeration areas, representing 74 districts and Kampala Capital City Authority. Enumeration occurred from August 20 to September 12, 2014. In each enumeration area, 24 households were randomly selected; the head of the household provided details regarding any household deaths within the previous 12 months. Two household members were randomly selected for a head-to-toe verbal interview to determine existing untreated and treated surgical conditions., Results: In 2315 households, we surveyed 4248 individuals: 461 (10.6%) reported 1 or more conditions requiring at least surgical consultation [95% confidence interval (CI) 8.9%-12.4%]. The most frequent barrier to surgical care was the lack of financial resources for the direct cost of care. Of the 153 household deaths recalled, 53 deaths (34.2%; 95% CI 22.1%-46.3%) were associated with surgically treatable signs/symptoms. Shortage of time was the most frequently cited reason (25.8%) among the 11.6% household deaths that should have, but did not, receive surgical care (95% CI 6.4%-16.8%)., Conclusions: Unmet surgical need is prevalent in Uganda. There is an urgent need to expand the surgical care delivery system starting with the district-level hospitals. Routine surgical data collection at both the health facility and household level should be implemented.

Objective: According to recent estimates, at least 11% of the total global burden of disease is attributable to surgically-treatable diseases. In children, the burden is even more striking with up to 85% of children in low-income and middle-income countries (LMIC) having a surgically-treatable condition by age 15. Using population data from four countries, we estimated pediatric surgical needs amongst children residing in LMICs., Methods: A cluster randomized cross-sectional countrywide household survey (Surgeons OverSeas Assessment of Surgical Need) was done in four countries (Rwanda, Sierra Leone, Nepal and Uganda) and included demographics, a verbal head to toe examination, and questions on access to care. Global estimates regarding surgical need among children were derived from combined data, accounting for country-level clustering., Results: A total of 13,806 participants were surveyed and 6,361 (46.1%) were children (0-18 years of age) with median age of 8 (Interquartile range [IQR]: 4-13) years. Overall, 19% (1,181/6,361) of children had a surgical need and 62% (738/1,181) of these children had at least one unmet need. Based on these estimates, the number of children living with a surgical need in these four LMICs is estimated at 3.7 million (95% CI: 3.4, 4.0 million). The highest percentage of unmet surgical conditions included head, face, and neck conditions, followed by conditions in the extremities. Over a third of the untreated conditions were masses while the overwhelming majority of treated conditions in all countries were wounds or burns., Conclusion: Surgery has been elevated as an "indivisible, indispensable part of health care" in LMICs and the newly formed 2015 Sustainable Development Goals are noted as unachievable without the provision of surgical care. Given the large burden of pediatric surgical conditions in LMICs, scale-up of services for children is an essential component to improve pediatric health in LMICs.

Background: Globally, a staggering five billion people lack access to adequate surgical care. Sub-Saharan Africa represents one of the regions of greatest need. We sought to understand how geographic factors related to unmet surgical need (USN) in Uganda., Methods: We performed a geographic information system analysis of a nationwide survey on surgical conditions performed in 105 enumeration areas (EAs) representing the national population. At the district level, we determined the spatial autocorrelation of the following study variables: prevalence of USN, hub distance (distance from EA to the nearest surgical center), area of coverage (geographic catchment area of each center), tertiary facility transport time (average respondent-reported travel time), and care availability (rate of hospital beds by population and by district). We then used local indicators of spatial association (LISA) and spatial regression to identify any significant clustering of these study variables among the districts., Results: The survey enumerated 4248 individuals. The prevalence of USN varied from 2.0-45 %. The USN prevalence was highest in the Northern and Western Regions. Moran's I bivariate analysis indicated a positive correlation between USN and hub distance (p = 0.03), area of coverage (p = 0.02), and facility transport time (p = 0.03). These associations were consistent nationally. The LISA analysis showed a high degree of clustering among sets of districts in the Northern Sub-Region., Conclusions: This study demonstrates a statistically significant association between USN and the geographic variables examined. We have identified the Northern Sub-Region as the highest priority areas for financial investment to reduce this unmet surgical disease burden.

Purpose: Little is known about the prevalence of pediatric surgical conditions in low- and middle-income countries. Many children never seek medical care, thus the true prevalence of surgical conditions in children in Uganda is unknown. The objective of this study was to determine the prevalence of surgical conditions in children in Uganda., Methods: Using the Surgeons OverSeas Assessment of Surgical Need (SOSAS) survey, we enumerated 4248 individuals in 2315 households in 105 randomly selected clusters throughout Uganda. Children aged 0-18 were included if randomly selected from the household; for those who could not answer for themselves, parents served as surrogates., Results: Of 2176 children surveyed, 160 (7.4 %) reported a currently untreated surgical condition. Lifetime prevalence of surgical conditions was 14.0 % (305/2176). The predominant cause of surgical conditions was trauma (48.4 %), followed by wounds (19.7 %), acquired deformities (16.2 %), and burns (12.5 %). Of 90 pediatric household deaths, 31.1 % were associated with a surgically treatable proximate cause of death (28/90 deaths)., Conclusion: Although some trauma-related surgical burden among children can be adequately addressed at district hospitals, the need for diagnostics, human resources, and curative services for more severe trauma cases, congenital deformities, and masses outweighs the current capacity of hospitals and trained pediatric surgeons in Uganda., Competing Interests: Compliance with ethical standards Funding source Funding was provided by the Duke Global Health Institute, Duke University Department of Neurosurgery, University of Minnesota Department of Surgery, Makerere College of Health Sciences, and Johnson and Johnson Family of Companies. Funding sources played no role in study design, data collection, data analysis, or writing of the manuscript. All authors had full access to the data and had final responsibility for the decision to submit for publication.

Introduction: The first step in improving surgical care delivery in low- and middle-income countries (LMICs) is quantifying surgical need. The Surgeons OverSeas Assessment of Surgical Need (SOSAS) is a validated household survey that has been previously implemented in three LMICs with great success. We implemented the SOSAS survey in Uganda, a medium-sized country with comparatively more language and ethnic group diversity., Methods: The investigators partnered with the Performance Monitoring and Accountability 2020 (PMA2020) Uganda to access a data collection platform sampling 2520 households in 105 randomly selected enumeration areas. Due to geographic size consideration and language diversity, SOSAS's methodology was updated in three significant dimensions (1) technology, (2) staff management, and (3) questionnaire adaptations., Results: The SOSAS survey was successfully implemented with non-medically trained but field proven research assistants. We sampled 2315 of 2402 eligible households (response rate 96.4 %) and 4248 of 4374 eligible individual respondents (response rate 97.1 %). The female-to-male ratio was 51.1-48.9 %. Total survey cost was USD 73,145 and data collection occurred in 14 days., Discussion: SOSAS Uganda has demonstrated that non-medically trained, but university-educated, experienced researchers supervised by academic surgeons can successfully perform accurate data collection of SOSAS. SOSAS can be successfully implemented within larger and more diverse LMICs using existing national survey platforms, and SOSAS Uganda provides insights on how SOSAS can be executed specifically within other PMA2020 program countries.

The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (∼10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for two alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown., (© 2015 The Authors Protein Science published by Wiley Periodicals, Inc.)

Background: Noncommunicable diseases, including those requiring surgical care, are increasingly straining low- and middle-income countries. Globally, 11% of all disability-adjusted life-years lost result from conditions requiring surgery; however, little is known about country-specific burden. We piloted a household-based survey in a periurban district of Uganda to estimate the prevalence of surgical conditions and to identify logistical challenges., Methods: Our sample comprised 57 households in 5 enumeration areas in the Wakiso District, in central Uganda. Our survey tool was the Surgeons OverSeas Assessment of Surgical need. A household representative completed demographic and household death information, and 2 randomly selected household members completed questions on surgical conditions., Results: Of 96 participants, 6 (6.3%; 95% CI, 2.3-13.1) had an existing, untreated surgical condition. The lifetime prevalence of surgical conditions was 26% (25/96). The most common barrier to access to care was lack of financial resources. Of the 3 deaths reported, 2 were associated with surgery. The mean household interview time was 36 minutes. The greatest challenge was efficient coordination with local team members and government officials., Conclusion: In this setting, the current prevalence of surgical conditions was nearly 1 in 10 persons, and lifetime occurrence was high, at 1 in 4 persons. Addressable challenges led to question revisions and a change in the data collection platform. A full-country study is both feasible and necessary to characterize the met and unmet need for surgical care in Uganda., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: A safe and adequate supply of blood is critical to improving health care systems in sub-Saharan Africa, where little is known about the current use of blood. The aim of this study was to comprehensively describe the use of blood at a tertiary care hospital to inform future efforts to strengthen blood programs in resource-limited settings., Study Design and Methods: Data were collected from blood bank documentation for all units issued at Mulago Hospital Complex in Kampala, Uganda, from mid-January to mid-April 2014., Results: A total of 6330 units (69% whole blood, 32% red blood cells, 6% platelets, 2% plasma) were issued over the 3-month study period to 3662 unique patients. Transfusion recipients were 58% female and median age was 27 years (interquartile range [IQR], 14-41). Median pretransfusion hemoglobin was 5.6 g/dL (IQR, 4.0-7.2 g/dL, n = 1090). Strikingly, cancer was the top indication for transfusion (33.5%), followed by pregnancy-related complications (12.4%) and sickle cell disease (6.9%)., Conclusion: This study provides a comprehensive picture of blood use at a national referral hospital in sub-Saharan Africa. Noncommunicable diseases, particularly oncologic conditions, represent a large proportion of demand for transfusion services., (© 2015 AABB.)

Peptidoglycan (PG) is an extracytoplasmic glycopeptide matrix essential for the integrity of the envelope of most bacteria. The PG building block is a disaccharide-pentapeptide that is synthesized as a lipid-linked precursor called lipid II. The translocation of the amphipathic lipid II across the cytoplasmic membrane is required for subsequent incorporation of the disaccharide-pentapeptide into PG. In Escherichia coli, the essential inner membrane protein MurJ is the lipid II flippase. Previous studies showed that 8 charged residues in the central cavity region of MurJ are crucial for function. Here, we completed the functional analysis of all 57 charged residues in MurJ and demonstrated that the respective positive or negative charge of the 8 aforementioned residues is required for proper MurJ function. Loss of the negative charge in one of these residues, D39, causes a severe defect in MurJ biogenesis; by engineering an intragenic suppressor mutation that restores MurJ biogenesis, we found that this charge is also essential for MurJ function. Because of the low level of homology between MurJ and putative orthologs from Gram-positive bacteria, we explored the conservation of these 8 charged residues in YtgP, a homolog from Streptococcus pyogenes. We found that only 3 positive charges are similarly positioned and essential in YtgP; YtgP possesses additional charged residues within its predicted cavity that are essential for function and conserved among Gram-positive bacteria. From these data, we hypothesize that some charged residues in the cavity region of MurJ homologs are required for interaction with lipid II and/or energy coupling during transport., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Peptidoglycan (PG) is a polysaccharide matrix that protects bacteria from osmotic lysis. Inhibition of its biogenesis is a proven strategy for killing bacteria with antibiotics. The assembly of PG requires disaccharide-pentapeptide building blocks attached to a polyisoprene lipid carrier called lipid II. Although the stages of lipid II synthesis are known, the identity of the essential flippase that translocates it across the cytoplasmic membrane for PG polymerization is unclear. We developed an assay for lipid II flippase activity and used a chemical genetic strategy to rapidly and specifically block flippase function. We combined these approaches to demonstrate that MurJ is the lipid II flippase in Escherichia coli., (Copyright © 2014, American Association for the Advancement of Science.)

Cryptococcal meningitis is common in sub-Saharan Africa. Given the need for data for a rapid, point-of-care cryptococcal antigen (CRAG) lateral flow immunochromatographic assay (LFA), we assessed diagnostic performance of cerebrospinal fluid (CSF) culture, CRAG latex agglutination, India ink microscopy, and CRAG LFA for 832 HIV-infected persons with suspected meningitis during 2006-2009 (n = 299) in Uganda and during 2010-2012 (n = 533) in Uganda and South Africa. CRAG LFA had the best performance (sensitivity 99.3%, specificity 99.1%). Culture sensitivity was dependent on CSF volume (82.4% for 10 μL, 94.2% for 100 μL). CRAG latex agglutination test sensitivity (97.0%-97.8%) and specificity (85.9%-100%) varied between manufacturers. India ink microscopy was 86% sensitive. Laser thermal contrast had 92% accuracy (R = 0.91, p<0.001) in quantifying CRAG titers from 1 LFA strip to within <1.5 dilutions of actual CRAG titers. CRAG LFA is a major advance for meningitis diagnostics in resource-limited settings.

Gram-negative bacteria such as Escherichia coli build a peptidoglycan (PG) cell wall in their periplasm using the precursor known as lipid II. Lipid II is a large amphipathic molecule composed of undecaprenyl diphosphate and a disaccharide-pentapeptide that PG-synthesizing enzymes use to build the PG sacculus. During PG biosynthesis, lipid II is synthesized at the cytoplasmic face of the inner membrane and then flipped across the membrane. This translocation of lipid II must be assisted by flippases thought to shield the disaccharide-pentapeptide as it crosses the hydrophobic core of the membrane. The inner membrane protein MurJ is essential for PG biogenesis and homologous to known and putative flippases of the MOP (multidrug/oligo-saccharidyl-lipid/polysaccharide) exporter superfamily, which includes flippases that translocate undecaprenyl diphosphate-linked oligosaccharides across the cytoplasmic membranes of bacteria. Consequently, MurJ has been proposed to function as the lipid II flippase in E. coli. Here, we present a three-dimensional structural model of MurJ generated by the I-TASSER server that suggests that MurJ contains a solvent-exposed cavity within the plane of the membrane. Using in vivo topological studies, we demonstrate that MurJ has 14 transmembrane domains and validate features of the MurJ structural model, including the presence of a solvent-exposed cavity within its transmembrane region. Furthermore, we present functional studies demonstrating that specific charged residues localized in the central cavity are essential for function. Together, our studies support the structural homology of MurJ to MOP exporter proteins, suggesting that MurJ might function as an essential transporter in PG biosynthesis.

Overexpression or mutation of α-Synuclein is associated with protein aggregation and interferes with a number of cellular processes, including mitochondrial integrity and function. We used a whole-genome screen in the fruit fly Drosophila melanogaster to search for novel genetic modifiers of human [A53T]α-Synuclein-induced neurotoxicity. Decreased expression of the mitochondrial chaperone protein tumor necrosis factor receptor associated protein-1 (TRAP1) was found to enhance age-dependent loss of fly head dopamine (DA) and DA neuron number resulting from [A53T]α-Synuclein expression. In addition, decreased TRAP1 expression in [A53T]α-Synuclein-expressing flies resulted in enhanced loss of climbing ability and sensitivity to oxidative stress. Overexpression of human TRAP1 was able to rescue these phenotypes. Similarly, human TRAP1 overexpression in rat primary cortical neurons rescued [A53T]α-Synuclein-induced sensitivity to rotenone treatment. In human (non)neuronal cell lines, small interfering RNA directed against TRAP1 enhanced [A53T]α-Synuclein-induced sensitivity to oxidative stress treatment. [A53T]α-Synuclein directly interfered with mitochondrial function, as its expression reduced Complex I activity in HEK293 cells. These effects were blocked by TRAP1 overexpression. Moreover, TRAP1 was able to prevent alteration in mitochondrial morphology caused by [A53T]α-Synuclein overexpression in human SH-SY5Y cells. These results indicate that [A53T]α-Synuclein toxicity is intimately connected to mitochondrial dysfunction and that toxicity reduction in fly and rat primary neurons and human cell lines can be achieved using overexpression of the mitochondrial chaperone TRAP1. Interestingly, TRAP1 has previously been shown to be phosphorylated by the serine/threonine kinase PINK1, thus providing a potential link of PINK1 via TRAP1 to α-Synuclein., Competing Interests: The authors have declared that no competing interests exist.

Data presented previously as an abstract at the 2011 CUGH Global Health Conference in Montreal, Canada on 15 Nov 2011. The long-term survival of HIV-infected persons with symptomatic cryptococcal meningitis and asymptomatic, subclinical cryptococcal antigenemia (CRAG+) is unknown. We prospectively enrolled 25 asymptomatic, antiretroviral therapy (ART)-naïve CRAG+ Ugandans with CD4<100 cells/mcL who received pre-emptive fluconazole treatment (CRAG+ cohort) and 189 ART-naïve Ugandans with symptomatic cryptococcal meningitis treated with amphotericin (CM cohort). The 10-week survival was 84% (95%CI: 70-98%) in the CRAG+ cohort and 57% (95%CI: 50%-64%) in the CM cohort. The CRAG+ cohort had improved five-year survival of 76% (95%CI: 59%-93%) compared to 42% (95%CI: 35%-50%) in the CM cohort (P = 0.001). The two cohorts had similar immunosuppression pre-ART with median CD4 counts of 15 vs. 21 CD4/mcL in the CRAG+ and CM cohorts, respectively (P = 0.45). Despite substantial early mortality, subsequent 5-year survival of persons surviving 6-months was excellent (>88%), demonstrating that long term survival is possible in resource-limited settings. Pre-ART CRAG screening with preemptive fluconazole treatment and improved CM treatment(s) are needed to reduce AIDS-attributable mortality due to cryptococcosis which remains 20-25% in sub-Saharan Africa.

Infection and disease associated with Neisseria gonorrhoeae, the gonococcus, continue to be a global health problem. Asymptomatic and subclinical gonococcal infections occur at a high frequency in females; thus, the true incidence of N. gonorrhoeae infections are presumed to be severely underestimated. Inherent to this asymptomatic/subclinical diseased state is the continued prevalence of this organism within the general population, as well as the medical, economic, and social burden equated with the observed chronic, disease sequelae. As infections of the lower female genital tract (i.e., the uterine cervix) commonly result in subclinical disease, it follows that the pathobiology of cervical gonorrhea would differ from that observed for other sites of infection. In this regard, the potential responses to infection that are generated by the female reproductive tract mucosa are unique in that they are governed, in part, by cyclic fluctuations in steroid hormone levels. The lower female genital tract has the further distinction of being able to functionally discriminate between resident commensal microbiota and transient pathogens. The expression of functionally active complement receptor 3 by the lower, but not the upper, female genital tract mucosa; together with data indicating that gonococcal adherence to and invasion of primary cervical epithelial cells and tissue are predominately aided by this surface-expressed host molecule; provide one explanation for asymptomatic/subclinical gonococcal cervicitis. However, co-evolution of the gonococcus with its sole human host has endowed this organism with variable survival strategies that not only aid these bacteria in successfully evasion of immune detection and function but also enhance cervical colonization and cellular invasion. To this end, we herein summarize current knowledge pertaining to the pathobiology of gonococcal infection of the human cervix.

Staphylococcus aureus encodes the Sec-independent Ess secretion pathway, an ortholog of mycobacterial T7 secretion systems which is required for the virulence of this Gram-positive microbe. The Ess (ESX secretion) pathway was previously defined as a genomic cluster of eight genes, esxA, esaA, essA, essB, esaB, essC, esaC, and esxB. essABC encode membrane proteins involved in the stable expression of esxA, esxB, and esaC, genes specifying three secreted polypeptide substrates. esaB, which encodes a small cytoplasmic protein, represses the synthesis of EsaC but not that of EsxA and EsxB. Here we investigated a hitherto uncharacterized gene, esaD, located downstream of esxB. Expression of esaD is activated by mutations in esaB and essB. EsaD, the 617-amino-acid product of esaD, is positioned in the membrane and is also accessible to EsaD-specific antibodies on the bacterial surface. S. aureus mutants lacking esaD are defective in the secretion of EsxA. Following intravenous inoculation of mice, S. aureus esaD mutants generate fewer abscesses with a reduced bacterial load compared to wild-type parent strain Newman. The chromosomes of Listeria and Bacillus species with Ess pathways also harbor esaD homologues downstream of esxB, suggesting that the contributory role of EsaD in Ess secretion may be shared among Gram-positive pathogens.

We have recorded laser excitation spectra of transitions from the ground X(1)Sigma(+) state of gaseous gold fluoride (AuF) into three excited electronic states in the visible region. We prepared the sample in a dc electric discharge by flowing a dilute mixture of SF(6) in argon through a hollow gold cathode. Two of these electronic states give rise to the previously reported yellow bands of the molecule, for which a rotational analysis is given here for the first time. We have analyzed the (0,0), (1,1), (0,1), and (1,2) bands of these two transitions, which we identify as [17.8]0(+)-X(1)Sigma(+) and [17.7]1-X(1)Sigma(+); their red-degraded (0,0) band heads lie at 563.0 and 566.2 nm, respectively. The (0,0) band of a new, red-degraded [14.0]1-X(1)Sigma(+) transition at 715.1 nm has also been recorded and analyzed. An accurate set of molecular constants of the three excited states as well as the ground state has been determined by least-squares fitting all of the optical data together with measurements made by other workers of the pure rotational spectrum of AuF in its ground state. These constants include the electronic term energies, vibrational frequencies, rotational constants, and Omega-doubling constants. We discuss the nature of these three excited electronic states in terms of the ionic Au(+)F(-) electronic configurations from which they are derived.

The [17.7]1-X(1)Sigma(+) (0,0) band of AuF at 566 nm has been studied by laser excitation spectroscopy. The molecule was prepared in a dc electric discharge by flowing a dilute mixture of SF(6) in argon through a hollow gold cathode. The rotational structure of the band has been analyzed for the first time, yielding accurate values for the rotational and Omega-type doubling constants of the upper state. Hyperfine splittings arising from both the (197)Au and (19)F nuclei have been resolved by recording the spectrum at sub-Doppler resolution using the technique of intermodulated fluorescence spectroscopy. The hyperfine structure is dominated by the (197)Au magnetic dipole interaction in the [17.7]1 state, with the (197)Au magnetic hyperfine constant determined to be h(1) = -543(4) MHz. It is demonstrated that the negative value of this constant implies that the [17.7]1 state has significant (3)Delta(1) character and that spin-orbit mixing with a (1)Pi(1) state may be providing the transition intensity to the ground electronic state.

Dyspnea in the patient with cardiopulmonary disease has been defined and discussed, emphasizing those conditions which increase the work of breathing: increased elastic resistance; increased airway obstruction; and deformities of the chest wall, lung, and pleura. Methods of assessing the cardiopulmonary status of the patient as it relates to each condition were presented. Determining and understanding such clinical signs as patterns of breathing, cough and secretions, râles, rhonchi, wheezes, diminished or augmented breath sounds, poor or unequal expansion of the rib cage, and dullness or hyperresonance on percusion is extremely important for the nurse in order to accurately and comprehensively make a nursing assessment. Such an assessment can provide the information necessary to plan more effectively for the care of the patient with cardiopulmonary disease.