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Introduction Post-COVID-19 condition, or syndrome, also known as long COVID, is an infection-associated chronic condition that can develop after a SARS-CoV-2 infection and last at least 3 months to years. Despite representing a high burden for the Unified Health System (SUS), which has affected millions of Brazilians, it has received limited attention in Brazil. Prevalence studies to date have failed to include a broad representation of the population, and there has been insufficient exploration of the impact on people’s lives and the burden of and barriers to accessing health services. This article presents the research protocol for the quantitative component of a mixed methods project to produce evidence to inform SUS’s provision of care for long COVID. The protocol was designed to study long COVID in SUS patients hospitalised for COVID-19 in a large city in Southeast Brazil to capture symptoms and factors associated with the syndrome, effects on quality of life and employment, health needs, use of health services and barriers to accessing necessary healthcare.Methods and analysis An ambidirectional cohort study to capture data retrospectively and prospectively from adults previously discharged from SUS hospitals for COVID-19. The study involves up to two telephone surveys with the patients or proxies selected from a sampling plan for population estimates. Survey questions include baseline and follow-up data on demographic, socioeconomic, comorbidities, work status, health-related quality of life, vaccination status, long COVID symptoms, healthcare needs, use and barriers to access. Descriptive and appropriate multivariable analyses will be employed.Ethics and dissemination The project was approved by the Research Ethics Committees of participant institutions and by the Brazilian National Research Ethics Commission. All participants provided verbal consent. We plan to publish articles in scientific journals and multimedia resources for SUS professionals and the general population.

Abstract Background The use of systems engineering tools, including the development and use of care cascades using routinely collected data, process mapping, and continuous quality improvement, is used for frontline healthcare workers to devise systems level change. South Africa experiences high rates of tuberculosis (TB) infection and disease as well as HIV co-infection. The Department of Health has made significant gains in HIV services over the last two decades, reaching their set “90–90-90” targets for HIV. However, TB services, although robust, have lagged in comparison for both disease and infection. The Systems Analysis and Improvement Approach (SAIA) is a five-step implementation science method, drawn from systems engineering, to identify, define, and implement workflow modifications using cascade analysis, process mapping, and repeated quality improvement cycles within healthcare facilities. Methods This stepped-wedge cluster randomized trial will evaluate the effectiveness of SAIA on TB (SAIA-TB) cascade optimization for patients with TB and high-risk contacts across 16 clinics in four local municipalities in the Sarah Baartman district, Eastern Cape, South Africa. We hypothesize that SAIA-TB implementation will lead to a 20% increase in each of: TB screening, TB preventive treatment initiation, and TB disease treatment initiation during the 18-month intervention period. Focus group discussions and key informant interviews with clinic staff will also be conducted to determine drivers of implementation variability across clinics. Discussion This study has the potential to improve TB screening, treatment initiation, and completion for both active disease and preventive measures among individuals with and without HIV in a high burden setting. SAIA-TB provides frontline health care workers with a systems-level view of their care delivery system with the aim of sustainable systems-level improvements. Trial registration Clinicaltrials.gov, NCT06314386. Registered 18 March 2024, https://clinicaltrials.gov/study/NCT06314386 . NCT06314386.

Background Task-sharing of spinal anaesthesia care by non-specialist graduate physicians, termed medical officers (MOs), is commonly practised in rural Indian healthcare facilities to mitigate workforce constraints. We sought to assess whether spinal anaesthesia failure rates of MOs were non-inferior to those of consultant anaesthesiologists (CA) following a standardised educational curriculum.Methods We performed a randomised, non-inferiority trial in three rural hospitals in Tamil Nadu and Chhattisgarh, India. Patients aged over 18 years with low perioperative risk (ASA I & II) were randomised to receive MO or CA care. Prior to the trial, MOs underwent task-based anaesthesia training, inclusive of remotely accessed lectures, simulation-based training and directly observed anaesthetic procedures and intraoperative care. The primary outcome measure was spinal anaesthesia failure with a non-inferiority margin of 5%. Secondary outcome measures consisted of incidence of perioperative and postoperative complications.Findings Between 12 July 2019 and 8 June 2020, a total of 422 patients undergoing surgical procedures amenable to spinal anaesthesia care were randomised to receive either MO (231, 54.7%) or CA care (191, 45.2%). Spinal anaesthesia failure rate for MOs (7, 3.0%) was non-inferior to those of CA (5, 2.6%); difference in success rate of 0.4% (95% CI=0.36–0.43%; p=0.80). Additionally, there were no statistically significant differences observed between the two groups for intraoperative or postoperative complications, or patients’ experience of pain during the procedure.Interpretation This study demonstrates that failure rates of spinal anaesthesia care provided by trained MOs are non-inferior to care provided by CAs in low-risk surgical patients. This may support policy measures that use task-sharing as a means of expanding anaesthesia care capacity in rural Indian hospitals.Trial registration number NCT04438811.

Abstract Background In midwifery education, the clinical learning experience (CLE) is a critical component to gaining competency and should comprise greater than 50% of a student’s education. Many studies have identified positive and negative factors affecting students’ CLE. However, few studies have directly compared the difference in CLE based on placement at a community clinic versus a tertiary hospital. Methods The aim of this study was to examine how clinical placement site, clinic or hospital, impacts students’ CLE in Sierra Leone. A once 34-question survey was given to midwifery students attending one of four public midwifery schools in Sierra Leone. Median scores were compared for survey items by placement site using Wilcoxon tests. The relationship between clinical placement and student’s experience were assessed using multilevel logistic regression. Results Two-hundred students (hospitals students = 145 (72.5%); clinic students = 55 (27.5%) across Sierra Leone completed surveys. Most students (76%, n = 151) reported satisfaction with their clinical placement. Students placed at clinics were more satisfied with opportunities to practice/develop skills (p = 0.007) and more strongly agreed preceptors treated them with respect (p = 0.001), helped improve their skills (p = 0.001), provided a safe environment to ask questions (p = 0.002), and had stronger teaching/mentorship skills (p = 0.009) than hospital students. Students placed at hospitals had greater satisfaction in exposure to certain clinical opportunities including completing partographs (p

Objectives To estimate the economic impact of failure to find and treat tuberculosis disease and prevent tuberculosis infection from progressing to active disease.Design Estimating the economic cost of not finding and treating a patient suffering from tuberculosis.Setting Estimation methodology is developed in the Indian context, as informed by local costs and reported tuberculosis epidemiology.Participants No individual participants were included.Primary and secondary outcome measures The primary outcome measure is the total cost of patients with drug-susceptible and drug-resistant tuberculosis who are and are not found and treated by tuberculosis programmes, including costs for medications, lost productivity, healthcare services and furthered transmission. We calculate the economic burdens by varying the number of individuals a person sick with tuberculosis infects (10 or 15 people) and the risk of progression to tuberculosis disease if infected (5 or 8%). The secondary outcome measure is the amount saved by finding a patient early or who would not have otherwise been found. All costs are presented in US dollars (exchange rate: 72 Indian rupees/1 US$).Results By finding and treating a patient early before furthered transmission occurs—or stopping progression of tuberculosis infection to tuberculosis disease with preventive therapy—the Indian health system can save US$5502 to US$15 825 and US$5846 to US$25 575, for each individual with drug-susceptible and drug-resistant tuberculosis, respectively, across scenarios.Conclusions These estimates provide crude, lower bounds for the potential costs of not appropriately diagnosing and treating a single patient with active tuberculosis in a timely manner, or preventing a patient with tuberculosis infection from progressing to active disease. The actual financial burden on society is far higher than estimated using this simple, short-term cost-effective analyses. Our results highlight the limitations of tuberculosis costing models to date, and demonstrate the importance of accounting for airborne transmission of tuberculosis.

Abstract Background 7-Methylxanthine, a derivative of caffeine noted for its lack of toxicity and ability to treat and even prevent myopia progression, is a high-value biochemical with limited natural availability. Attempts to produce 7-methylxanthine through purely chemical methods of synthesis are faced with complicated chemical processes and/or the requirement of a variety of hazardous chemicals, resulting in low yields and racemic mixtures of products. In recent years, we have developed engineered microbial cells to produce several methylxanthines, including 3-methylxanthine, theobromine, and paraxanthine. The purpose of this study is to establish a more efficient biosynthetic process for the production of 7-methylxanthine from caffeine. Results Here, we describe the use of a mixed-culture system composed of Escherichia coli strains engineered as caffeine and theobromine “specialist” cells. Optimal reaction conditions for the maximal conversion of caffeine to 7-methylxanthine were determined to be equal concentrations of caffeine and theobromine specialist cells at an optical density (600 nm) of 50 reacted with 2.5 mM caffeine for 5 h. When scaled-up to 560 mL, the simple biocatalytic reaction produced 183.81 mg 7-methylxanthine from 238.38 mg caffeine under ambient conditions, an 85.6% molar conversion. Following HPLC purification and solvent evaporation, 153.3 mg of dried 7-methylxanthine powder was collected, resulting in an 83.4% product recovery. Conclusion We present the first report of a biocatalytic process designed specifically for the production and purification of the high-value biochemical 7-methylxanthine from caffeine using a mixed culture of E. coli strains. This process constitutes the most efficient method for the production of 7-methylxanthine from caffeine to date.

Abstract Mobile screening units can help close tuberculosis case detection gaps. Placing screening units where people at high risk for undiagnosed tuberculosis preferentially spend time could make screening more resource-effective. We conducted a case–control study in Lima, Peru to identify locations where people with tuberculosis were more likely to spend time than community controls. We surveyed participants about activity locations over the past 6 months. We used density-based clustering to assess how patient and control activity locations differed, and logistic regression to compare location-based exposures. We included 109 tuberculosis patients and 79 controls. In density-based clustering analysis, the two groups had similar patterns of living locations, but their work locations clustered in distinct areas. Both groups were similarly likely to use public transit, but patients predominantly used buses and were less likely to use rapid transit (adjusted odds ratio [aOR] 0.31, 95% confidence interval [CI] 0.10–0.96) or taxis (aOR 0.42, 95% CI 0.21–0.85). Patients were more likely to have spent time in prison (aOR 11.55, 95% CI 1.48–90.13). Placing mobile screening units at bus terminals serving locations where tuberculosis patients have worked and within and around prisons could help reach people with undiagnosed tuberculosis.

Objectives: Annually, more than 30% of individuals with tuberculosis (TB) remain undiagnosed. We aimed to assess whether geographic accessibility measures can identify neighborhoods that would benefit from TB screening services targeted toward closing the diagnosis gap. Methods: We used data from a community-based mobile TB screening program in Carabayllo district, Lima, Peru. We constructed four accessibility measures from the geographic center of neighborhoods to health facilities. We used logistic regression to assess the association between these measures and screening uptake in one's residential neighborhood versus elsewhere, with quasi-information criterion values to assess the association. Results: We analyzed the screening locations for 25,000 Carabayllo residents from 49 neighborhoods. Pedestrian walk time was preferable to Euclidean distance or vehicular time in our models. For each additional 12 minutes walking time between the neighborhood and the health facility, the odds of residents using TB screening units located in their neighborhoods increased by 50% (95% CI: 26%–78%). Females had 9% (95% CI: 3%–16%) increased odds versus males of using a screening unit in their own neighborhood. Conclusion: Placing mobile TB screening units in neighborhoods with longer pedestrian time to access health facilities could benefit individuals who face more acute access barriers to health care.

Abstract Background We assessed the feasibility of implementing psychological counseling services (PCS) for mothers of children with autism spectrum disorders (ASD) integrated within special education settings in urban Bangladesh. Method In two special education schools for ASD in Dhaka City, trained female psychologists screened mothers using the Patient Health Questionnaire (PHQ-9). PCS was administered to all the mothers irrespective of a diagnosis of depression. Mothers with a PHQ-9 score >4 who met criteria for a major depressive episode (MDE) based on the DSM-IV Structured Interview Axis I Disorders (SCID-I) were also administered skill-building training through monthly home visits to support ASD care. The level of depression was assessed by the Depression Measurement Scale (DMS), and quality of life (QoL) was measured by Visual Analogue Scale (VAS) of EQ5D5L scale before and after PCS. Result Among 188 mothers enrolled in the study, 81 (43%) received PCS, and 27.1% (22) had MDE. In the first month, 73 sessions were scheduled and 60 completed (85%). In the last month, 53 sessions were scheduled and 52 completed (98%). The mean DMS score decreased from 79.5 ± 23 to 60 ± 20 (p = 0.004), and DMS scores were significantly higher among mothers with MDE (97.8 ± 12.1 v. 69.9 ± 22.1; p < 0.001) compared to those without MDE (72.7 ± 22.6 v. 56.1 ± 18.1; p = 0.003). The mean VAS score improved from 70.3 ± 14.1 to 80.2 ± 13.3 (p = 0.001) between the first and the last session. Changes in DMS were negatively correlated with changes in VAS scores (β: −0.213, 95% CI 0.370 to −0.056). Conclusion Within special education schools for ASD in urban Bangladesh, it was feasible to administer an integrated program of PCS for mothers of children with ASD by trained psychologists who were able to screen and intervene to reduce their level of depression and improve their quality of life.

Abstract Tuberculosis screening programs commonly target areas with high case notification rates. However, this may exacerbate disparities by excluding areas that already face barriers to accessing diagnostic services. We compared historic case notification rates, demographic, and socioeconomic indicators as predictors of neighborhood-level tuberculosis screening yield during a mobile screening program in 74 neighborhoods in Lima, Peru. We used logistic regression and Classification and Regression Tree (CART) analysis to identify predictors of screening yield. During February 7, 2019–February 6, 2020, the program screened 29,619 people and diagnosed 147 tuberculosis cases. Historic case notification rate was not associated with screening yield in any analysis. In regression analysis, screening yield decreased as the percent of vehicle ownership increased (odds ratio [OR]: 0.76 per 10% increase in vehicle ownership; 95% confidence interval [CI]: 0.58–0.99). CART analysis identified the percent of blender ownership (≤ 83.1% vs > 83.1%; OR: 1.7; 95% CI: 1.2–2.6) and the percent of TB patients with a prior tuberculosis episode (> 10.6% vs ≤ 10.6%; OR: 3.6; 95% CI: 1.0–12.7) as optimal predictors of screening yield. Overall, socioeconomic indicators were better predictors of tuberculosis screening yield than historic case notification rates. Considering community-level socioeconomic characteristics could help identify high-yield locations for screening interventions.

Objectives To evaluate the feasibility of the Zero TB Indicator Framework as a tool for assessing the quality of tuberculosis (TB) case-finding, treatment and prevention services in Mongolia.Setting Primary health centres, TB dispensaries, and surrounding communities in four districts of Mongolia.Design Three retrospective cross-sectional cohort studies, and two longitudinal studies each individually nested in one of the cohort studies.Participants 15 947 community members from high TB-risk populations; 8518 patients screened for TB in primary health centres and referred to dispensaries; 857 patients with index TB and 2352 household contacts.Primary and secondary outcome measures 14 indicators of the quality of TB care defined by the Zero TB Indicator Framework and organised into three care cascades, evaluating community-based active case-finding, passive case-finding in health facilities and TB screening and prevention among close contacts; individual and health-system predictors of these indicators.Results The cumulative proportions of participants receiving guideline-adherent care varied widely, from 96% for community-based active case-finding, to 79% for TB preventive therapy among household contacts, to only 67% for passive case-finding in primary health centres and TB dispensaries (range: 29%–80% across districts). The odds of patients completing active TB treatment decreased substantially with increasing age (aOR: 0.76 per decade, 95% CI: 0.71 to 0.83, p

Objectives Though hospital leaders across the USA have invested significant resources in collection of patient-reported outcome measures (PROMs), there are very limited data on the impetus for hospital leadership to establish PROM programmes. In this qualitative study, we identify the drivers and motivators of PROM collection among hospital leaders in the USA.Design Exploratory qualitative study.Setting Thirty-seven hospital leaders representing seven different institutions with successful PROMs programs across twenty US states.Methods Semistructured interviews conducted with hospital leaders. Transcripts were analysed using thematic analysis.Results Leaders strongly believe that collecting PROMs is the ‘right thing to do’ and that the culture of the institution plays an important role in enabling PROMs. The study participants often believe that their institutions deliver superior care and that PROMs can be used to demonstrate the value of their services to payors and patients. Direct financial incentives are relatively weak motivators for collection of PROMs. Most hospital leaders have reservations about using PROMs in their current state as a meaningful performance metric.Conclusion These findings suggest that hospital leaders feel a strong moral imperative to collect PROMs, which is also supported by the culture of their institution. Although PROMs are used in negotiations with payors, direct financial return on investment is not a strong driver for the collection of PROMs. Understanding why leaders of major healthcare institutions invest in PROMs is critical to understanding the role that PROMs play in the US healthcare system.

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Objective: To demonstrate the utility of cold ischemia during robotic-assisted laparoscopic partial nephrectomy (RAPN). Patients and surgical procedure: Patients that are eligible for nephron-sparing surgery for management of renal neoplasms include those with solitary anatomical or functioning kidney, chronic kidney disease (CKD) and bilateral renal masses. The utilization of hilar clamping and resultant ischemia time is an inherent limitation of partial nephrectomy. We present a case of a large, complex unilateral renal mass in a patient with stage III CKD that required hilar clamping. In an effort to maximally preserve post-operative renal function, we performed RAPN utilizing cold ischemia for definitive surgical management. Finding an optimal method for laparoscopic ice delivery to induce ischemia has been challenging with many mechanisms proposed (Ramani et al., 2006). To date, a general consensus has not been reached on which method supersedes others. Results: Cold ischemia was accomplished using a modified 10 cc syringe to deliver 300 cc of ice through a dedicated 12 mm trocar. A clamp time of 32 min was necessary for tumor enucleation and renorraphy. Estimated blood loss was 200 cc and final pathology showed Fuhrman grade 3 clear cell RCC (pT1aNxM0) with negative margins. The patient was discharged after an uneventful hospitalization on post-operative day 3. At 2-week follow-up his renal function was comparable to preoperative values. Conclusions: Cold ischemia during RAPN is feasible and may facilitate prolonged ischemia time in the setting of a complex renal mass and the decision to pursue nephron-sparing surgery.

Abstract Background There is limited research to guide TB treatment specifically in pregnant women and few studies have described the presentation of TB in pregnant women. We aimed to understand TB presentation and treatment outcomes in pregnant women in a low HIV burden setting. We describe a cohort of women of childbearing age treated for TB disease in Lima, Peru, and compare clinical presentation and treatment outcomes among pregnant and non-pregnant women between 2009 and 2012, including 36 pregnant women. Methods This is a prospective cohort study. Subjects were recruited from across 106 public health centers in Lima, Peru. Baseline demographic, medical history, and drug-susceptibility test results were collected. We used descriptive statistics to describe demographic and clinical characteristics of the women using Pearson chi-squared, Fisher’s exact tests, or Kruskal-Wallis. Results Among 4500 individuals with pulmonary TB disease, 1334 women were included in analysis with 36 (2.69%) pregnant women. Pregnant women had similar demographics, past medical histories, and clinical presentation to non-pregnant women, except being more likely to be married (p = 0.01) and have cardiac disease (p = 0.04) and less likely to have weight loss (p = 0.05). Twenty (71.4%) pregnant women had pan-susceptible TB compared with 616 (63.1%) non-pregnant women; four (14.3%) pregnant women had mono-resistant TB compared with 154 (15.8%) non-pregnant women; and four (14.3%) pregnant women had multi-drug-resistant TB compared with 140 (14.3%) of non-pregnant women (p = 0.53). Twenty-eight (96.6%) pregnant women had a successful outcome (cure, completed treatment, treatment ended early by clinical team) while one (3.4%) had an unsuccessful outcome (treatment failed) and 1074 (97.3%) non-pregnant women had a successful outcome while 30 (2.7%) had an unsuccessful outcome (p = 0.56). Conclusion In this cohort with low HIV co-infection, we found high TB treatment success rates in both pregnant and non-pregnant women, irrespective of drug-susceptibility profiles. If treated appropriately, pregnant women with TB disease can have successful outcomes.

Abstract Background: Identification of evidence-based factors related to status of the clinical research professional (CRP) workforce at academic medical centers (AMCs) will provide context for National Center for Advancing Translational Science (NCATS) policy considerations and guidance. The objective of this study is to explore barriers and opportunities related to the recruitment and retention of the CRP workforce. Materials and Methods: Qualitative data from a series of Un-Meeting breakout sessions and open-text survey questions were analyzed to explore barriers and recommendations for improving AMC CRP recruitment, retention and diversity. Results: While certain institutions have established competency-based frameworks for job descriptions, standardization remains generally lacking across CTSAs. AMCs report substantial increases in unfilled CRP positions leading to operational instability. Data confirmed an urgent need for closing gaps in CRP workforce at AMCs, especially for attracting, training, retaining, and diversifying qualified personnel. Improved collaboration with human resource departments, engagement with principal investigators, and overcoming both organizational and resource challenges were suggested strategies, as well as development of outreach to universities, community colleges, and high schools raising awareness of CRP career pathways. Discussion: Based on input from 130 CRP leaders at 35 CTSAs, four National Institute of General Medical Sciences’ Institutional Development Award (IDeA) program sites, along with industry and government representatives, we identified several barriers to successful recruitment and retention of a highly trained and diverse CRP workforce. Results, including securing institutional support, champions, standardizing and adopting proven national models, improving local institutional policies to facilitate CRP hiring and job progression point to potential solutions.

Objective To apply a cascade-of-care framework to evaluate the effectiveness—by age of the child—of an intensified tuberculosis patient-finding intervention. Design From a prospective screening program at four hospitals in Pakistan (2014–2016) we constructed a care cascade comprising six steps: screened, positive screen, evaluated, diagnosed, started treatment, and successful outcome. We evaluated the cascade by each year of age from 0 to 14 and report the age-specific mean proportion and standard deviation. Results On average across all ages, only 12.5% (standard deviation: 2.0%) of children with a positive screen were not evaluated. Among children who had a complete evaluation, the highest percentages of children diagnosed with tuberculosis were observed in children 0–4 (mean: 31.9%; standard deviation: 4.8%), followed by lower percentages in children 5–9 (mean: 22.4%; standard deviation: 2.2%), and 10–14 (mean: 26.0%; standard deviation:5.4%). Nearly all children diagnosed with tuberculosis initiated treatment, and an average of 93.3% (standard deviation: 3.3%) across all ages had successful treatment outcomes. Conclusions This intervention was highly effective across ages 0–14 years. Our study illustrates the utility of applying operational analyses of age-stratified cascades to identify age-specific gaps in pediatric tuberculosis care that can guide future, novel interventions to close these gaps.

ObjectiveTo use routinely collected data, with the addition of geographic information and census data, to identify local hot spots of rates of reported tuberculosis cases.DesignResidential locations of tuberculosis cases identified from eight public health facilities in Lima, Peru (2013-2018) were linked to census data to calculate neighborhood-level annual case rates. Heat maps of tuberculosis case rates by neighborhood were created. Local indicators of spatial autocorrelation, Moran's I, were used to identify where in the study area spatial clusters and outliers of tuberculosis case rates were occurring. Age- and sex-stratified case rates were also assessed.ResultsWe identified reports of 1,295 TB cases across 74 neighborhoods during the five-year study period, for an average annual rate of 124.2 reported TB cases per 100,000 population. In evaluating case rates by individual neighborhood, we identified a median rate of reported cases of 123.6 and a range from 0 to 800 cases per 100,000 population. Individuals aged 15-44 years old and men had higher case rates than other age groups and women. Locations of both hot and cold spots overlapped across age- and gender-specific maps.ConclusionsThere is significant geographic heterogeneity in rates of reported TB cases and evident hot and cold spots within the study area. Characterization of the spatial distribution of these rates and local hot spots may be one practical tool to inform the work of local coalitions to target TB interventions in their zones.

Aquatic surveys for fish in large water bodies (e.g., Laurentian Great Lakes of North America) often require a flexible approach using multiple methods, surveying different depths, and sampling across seasons, especially when the target species is elusive in its natural habitat. The round goby (Neogobius melanostomus) is an invasive, bottom-dwelling fish inhabiting rocky areas of all five Great Lakes. While trawl surveys are typically used for abundance assessments, angling has been demonstrated as a means of supplementing surveys with additional data. Yet, round goby abundance and distribution is still not well described. Recently, with considerable success, scientists have explored sampling environmental DNA (eDNA) to complement traditional monitoring techniques for population abundance estimates, early detection of invasive species, and spawning or migration events. Therefore, we collected eDNA from water samples alongside bottom trawls and hook and line angling in Lakes Huron and Michigan to detect round goby. eDNA samples were analyzed by both droplet digital PCR (ddPCR) and quantitative PCR (qPCR) to maximize the likelihood of detection. Overall, round goby was captured in 23% of the trawls, but the eDNA based methods detected round goby in 74% and 66% of samples by ddPCR and qPCR, respectively, mostly in samples collected at

ABSTRACT Adaptation of group A Streptococcus (GAS) to its human host is mediated by two-component systems that transduce external stimuli to regulate bacterial physiology. Among such systems, CsrRS (also known as CovRS) is the most extensively characterized for its role in regulating ∼10% of the GAS genome, including several virulence genes. Here, we show that extracellular magnesium and the human antimicrobial peptide LL-37 have opposing effects on the phosphorylation of the response regulator CsrR by the receptor kinase CsrS. Genetic inactivation of CsrS phosphatase or kinase activity, respectively, had similar but more pronounced effects on CsrR phosphorylation compared to growth in magnesium or LL-37. These changes in CsrR phosphorylation were correlated with the repression or activation of CsrR-regulated genes as assessed by NanoString analysis. Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) revealed CsrR occupancy at CsrRS-regulated promoters and lower-affinity associations at many other locations on the GAS chromosome. Because ChIP-seq did not detect CsrR occupancy at promoters associated with some CsrR-regulated genes, we investigated whether these genes might be controlled indirectly by intermediate regulators whose expression is modulated by CsrR. Transcriptional profiling of mutant strains deficient in the expression of either of two previously uncharacterized transcription regulators in the CsrR regulon indicated that one or both proteins participated in the regulation of 22 of the 42 CsrR-regulated promoters for which no CsrR association was detected by ChIP-seq. Taken together, these results illuminate CsrRS-mediated regulation of GAS gene expression through modulation of CsrR phosphorylation, CsrR association with regulated promoters, and the control of intermediate transcription regulators. IMPORTANCE Group A Streptococcus (GAS) is an important public health threat as a cause of sore throat, skin infections, life-threatening invasive infections, and the postinfectious complications of acute rheumatic fever, a leading cause of acquired heart disease. This work characterizes CsrRS, a GAS system for the detection of environmental signals that enables adaptation of the bacteria for survival in the human throat by regulating the production of products that allow the bacteria to resist clearance by the human immune system. CsrRS consists of two proteins: CsrS, which is on the bacterial surface to detect specific stimuli, and CsrR, which receives signals from CsrS and, in response, represses or activates the expression of genes coding for proteins that enhance bacterial survival. Some of the genes regulated by CsrR encode proteins that are themselves regulators of gene expression, thereby creating a regulatory cascade.

Water clarity is often the primary guiding factor in determining whether a prefiltration step is needed to increase volumes processed for a range of microbial endpoints. In this study, we evaluate the effect of filter pore size on the bacterial communities detected by 16S rRNA gene sequencing and incidence of two host-specific microbial source tracking (MST) markers in a range of coastal waters from southern Lake Michigan, using two independent data sets collected in 2015 (bacterial communities) and 2016–2017 (MST markers). Water samples were collected from river, shoreline, and offshore areas. For bacterial communities, each sample was filtered through a 5.0-μm filter, followed by filtration through a 0.22-μm filter, resulting in 70 and 143 filter pairs for bacterial communities and MST markers, respectively. Following DNA extraction, the bacterial communities were compared using 16S rRNA gene amplicons of the V3–V4 region sequenced on a MiSeq Illumina platform. Presence of human (Bacteroides HF183) and gull (Gull2, Catellicoccus marimammalium) host-specific MST markers were detected by qPCR. Actinobacteriota, Bacteroidota, and Proteobacteria, collectively represented 96.9% and 93.9% of the relative proportion of all phyla in the 0.22- and 5.0-μm pore size filters, respectively. There were more families detected in the 5.0-μm pore size filter (368) than the 0.22-μm (228). There were significant differences in the number of taxa between the two filter sizes at all levels of taxonomic classification according to linear discriminant analysis (LDA) effect size (LEfSe) with as many as 986 taxa from both filter sizes at LDA effect sizes greater than 2.0. Overall, the Gull2 marker was found in higher abundance on the 5.0-μm filter than 0.22 μm with the reverse pattern for the HF183 marker. This discrepancy could lead to problems with identifying microbial sources of contamination. Collectively, these results highlight the importance of analyzing pre- and final filters for a wide range of microbial endpoints, including host-specific MST markers routinely used in water quality monitoring programs. Analysis of both filters may increase costs but provides more complete genomic data via increased sample volume for characterizing microbial communities in coastal waters.

Objectives Identify barriers and facilitators to integrating community tuberculosis screening with mobile X-ray units into a health system.Methods Reach, effectiveness, adoption, implementation and maintenance evaluation.Setting 3-district region of Lima, Peru.Participants 63 899 people attended the mobile units from 7 February 2019 to 6 February 2020.Interventions Participants were screened by chest radiography, which was scored for abnormality by computer-aided detection. People with abnormal X-rays were evaluated clinically and by GeneXpert MTB/RIF (Xpert) sputum testing. People diagnosed with tuberculosis at the mobile unit were accompanied to health facilities for treatment initiation.Primary and secondary outcome measures Reach was defined as the percentage of the population of the three-district region that attended the mobile units. Effectiveness was defined as the change in tuberculosis case notifications over a historical baseline. Key implementation fidelity indicators were the percentages of people who had chest radiography performed, were evaluated clinically, had sputum samples collected, had valid Xpert results and initiated treatment.Results The intervention reached 6% of the target population and was associated with an 11% (95% CI 6 to 16) increase in quarterly case notifications, adjusting for the increasing trend in notifications over the previous 3 years. Implementation indicators for screening, sputum collection and Xpert testing procedures all exceeded 85%. Only 82% of people diagnosed with tuberculosis at the mobile units received treatment; people with negative or trace Xpert results were less likely to receive treatment. Suboptimal treatment initiation was driven by health facility doctors’ lack of familiarity with Xpert and lack of confidence in diagnoses made at the mobile unit.Conclusion Mobile X-ray units were a feasible and effective strategy to extend tuberculosis diagnostic services into communities and improve early case detection. Effective deployment however requires advance coordination among stakeholders and targeted provider training to ensure that people diagnosed with tuberculosis by new modalities receive prompt treatment.

Abstract Background Multidrug-resistant tuberculosis (MDR-TB) cohorts often lack long-term survival data, and are summarized instead by initial treatment outcomes. When using Cox proportional hazards models to analyze these cohorts, this leads to censoring subjects at the time of the initial treatment outcome, instead of them providing full survival data. This may violate the non-informative censoring assumption of the model and may produce biased effect estimates. To address this problem, we develop a tool to predict vital status at the end of a cohort period using the initial treatment outcome and assess its ability to reduce bias in treatment effect estimates. Methods We derive and apply a logistic regression model to predict vital status at the end of the cohort period and modify the unobserved survival outcomes to better match the predicted survival experience of study subjects. We compare hazard ratio estimates for effect of an aggressive treatment regimen from Cox proportional hazards models using time to initial treatment outcome, predicted vital status, and true vital status at the end of the cohort period. Results Models fit from initial treatment outcomes underestimate treatment effects by up to 22.1%, while using predicted vital status reduced this bias by 5.4%. Models utilizing the predicted vital status produce effect estimates consistently stronger and closer to the true treatment effect than estimates produced by models using the initial treatment outcome. Conclusions Although studies often use initial treatment outcomes to estimate treatment effects, this may violate the non-informative censoring assumption of the Cox proportional hazards model and result in biased treatment effect estimates. Using predicted vital status at the end of the cohort period may reduce this bias in the analyses of MDR-TB treatment cohorts, yielding more accurate, and likely larger, treatment effect estimates. Further, these larger effect sizes can have downstream impacts on future study design by increasing power and reducing sample size needs.

The coffee berry borer, the most economically important insect pest of coffee worldwide, is the only insect capable of feeding and reproducing solely on the coffee seed, a food source containing the purine alkaloid caffeine. Twenty-one bacterial species associated with coffee berry borers from Hawai’i, Mexico, or a laboratory colony in Maryland (Acinetobacter sp. S40, S54, S55, Bacillus aryabhattai, Delftia lacustris, Erwinia sp. S38, S43, S63, Klebsiella oxytoca, Ochrobactrum sp. S45, S46, Pantoea sp. S61, Pseudomonas aeruginosa, P. parafulva, and Pseudomonas sp. S30, S31, S32, S37, S44, S60, S75) were found to have at least one of five caffeine N-demethylation genes (ndmA, ndmB, ndmC, ndmD, ndmE), with Pseudomonas spp. S31, S32, S37, S60 and P. parafulva having the full complement of these genes. Some of the bacteria carrying the ndm genes were detected in eggs, suggesting possible vertical transmission, while presence of caffeine-degrading bacteria in frass, e.g., P. parafulva (ndmABCDE) and Bacillus aryabhattai (ndmA) could result in horizontal transmission to all insect life stages. Thirty-five bacterial species associated with the insect (Acinetobacter sp. S40, S54, S55, B. aryabhattai, B. cereus group, Bacillus sp. S29, S70, S71, S72, S73, D. lacustris, Erwinia sp. S38, S43, S59, S63, K. oxytoca, Kosakonia cowanii, Ochrobactrum sp. S45, S46, Paenibacillus sp. S28, Pantoea sp. S61, S62, P. aeruginosa, P. parafulva, Pseudomonas sp. S30, S31, S32, S37, S44, S60, S75, Stenotrophomonas sp. S39, S41, S48, S49) might contribute to caffeine breakdown using the C-8 oxidation pathway, based on presence of genes required for this pathway. It is possible that caffeine-degrading bacteria associated with the coffee berry borer originated as epiphytes and endophytes in the coffee plant microbiota.

Introduction: Depression and anxiety among tuberculosis (TB) patients can adversely affect TB treatment adherence and completion. Aim: We studied whether integrating mental health services into existing TB treatment programs would reduce symptoms of depression and anxiety and improve treatment completion among patients with drug-susceptible TB. Methods: Integrated practice units (IPUs) for TB and mental health were established within six existing TB treatment facilities in Karachi, Pakistan. Patients were screened for depression and anxiety and, if symptomatic, offered a mental health intervention consisting of at least four counseling sessions. We measured changes in reported levels of depression and anxiety symptoms from baseline following completion of counseling sessions, and rates of TB treatment completion. Results: Between February 2017 and June 2018, 3500 TB patients were screened for depression and anxiety. 1057 (30.2%) symptomatic patients received a baseline adherence session. 1012 enrolled for a mental health intervention received at least 1 counseling session. 522 (51.5%) reported no symptoms after four to six sessions. Symptomatic patients who completed at least four counseling sessions had higher rates of TB treatment completion than those who did not (92.9% vs 75.1%; p < 0.0001). Conclusion: Mental health interventions integrated within TB programs can help reduce symptoms of depression and anxiety and improve TB treatment completion.

PurposeTo evaluate the diagnostic value of symptoms used by daycares and schools to screen children and adolescents for SARS-CoV-2 infection, we analyzed data from a primary care setting.MethodsThis cohort study included all patients ≤17 years old who were evaluated at Providence Community Health Centers (PCHC; Providence, U.S.), for COVID-19 symptoms and/or exposure, and received SARS-CoV-2 polymerase chain reaction (PCR) testing between March-June 2020. Participants were identified from PCHC electronic medical records. For three age groups- 0-4, 5-11, and 12-17 years-we estimated the sensitivity, specificity, and area under the receiver operating curve (AUC) of individual symptoms and three symptom combinations: a case definition published by the Rhode Island Department of Health (RIDOH), and two novel combinations generated by different statistical approaches to maximize sensitivity, specificity, and AUC. We evaluated symptom combinations both with and without consideration of COVID-19 exposure. Myalgia, headache, sore throat, abdominal pain, nausea, anosmia, and ageusia were not assessed in 0-4 year-olds due to the lower reliability of these symptoms in this group.ResultsOf 555 participants, 217 (39.1%) were SARS-CoV-2-infected. Fever was more common among 0-4 years-olds (p = 0.002); older children more frequently reported fatigue (p = 0.02). In children ≥5 years old, anosmia or ageusia had 94-98% specificity. In all ages, exposure history most accurately predicted infection. With respect to individual symptoms, cough most accurately predicted infection in 95% but specificity ConclusionsAnosmia or ageusia in children ≥5 years old should raise providers' index of suspicion for COVID-19. However, our overall findings underscore the limited diagnostic value of symptoms.

BackgroundTargeted testing and treatment of TB infection to prevent disease is a pillar of TB elimination. Despite recent global commitments to greatly expand access to preventive treatment for TB infection, there remains a lack of research on how best to expand preventive treatment programs in settings with high TB burdens.MethodsWe conducted implementation research in Lima, Peru, around a multifaceted intervention to deliver TB preventive treatment to close contacts of all ages, health care workers, and people in congregate settings. Key interventions included use of the interferon gamma release assay (IGRA), specialist support for generalist physicians at primary-level health facilities, and treatment support by community health workers. We applied a convergent mixed methods approach to evaluate feasibility and acceptability based on a care cascade framework.FindingsDuring April 2019-January 2020, we enrolled 1,002 household contacts, 148 non-household contacts, 107 residents and staff of congregate settings, and 357 health care workers. Cumulative completion of the TB preventive care cascade was 34% for contacts ConclusionsWe attempted to close the gap in TB preventive treatment in Peru by expanding preventive services to adult contacts and other risk groups. While suboptimal, care cascade completion for adult contacts was consistent with what has been observed in high-income settings. The major losses in the care cascade occurred in completing evaluations and having doctors prescribe preventive treatment.

Streptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal glomerulonephritis. Genetic manipulation of this important pathogen is complicated by resistance of the organism to genetic transformation. Very low transformation efficiency is attributed to recognition and degradation of introduced foreign DNA by a type I restriction-modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten GAS strains that had been previously transformed with an unrelated plasmid revealed that six of the ten harbored a spontaneous mutation in hsdR, S, or M. The mutations were all different, and at least five of the six were predicted to result in loss of function of the respective hsd gene product. The unexpected occurrence of such mutations in previously transformed isolates suggested that the process of transformation selects for spontaneous inactivating mutations in the Hsd system. We investigated the possibility of exploiting the increased transformability of hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate representative of the globally dominant M1T1 clonal group. Mutant strain 854ΔhsdM exhibited a 5-fold increase in electrotransformation efficiency compared to the wild type parent strain and no obvious change in growth or off-target gene expression. We conclude that genetic transformation of GAS selects for spontaneous mutants in the hsdRSM restriction modification system. We propose that use of a defined hsdM mutant as a parent strain for genetic manipulation of GAS will enhance transformation efficiency and reduce the likelihood of selecting spontaneous hsd mutants with uncharacterized genotypes.

Team-based care has emerged as a promising strategy for primary care practices to provide high-quality care. We examine changes in patient experience of care and recommended cancer screening rates associated with a primary care transformation initiative that established team-based care. Our observational study included 13 academically affiliated primary care practices in the Boston, Massachusetts area that participated in 2 learning collaboratives: the first (2012-2014) aimed to establish team-based primary care, while the second (2014-2016) focused on improving patient safety and cancer screening. We identified 37 comparison practices of similar size and network affiliation. Using a difference-in-differences approach, we compared pre (2013) and post (2015) patient experience and recommended cancer screening rates between intervention and comparison practices. We estimated linear regression models, using inverse probability weighting to balance on observable differences. Massachusetts Health Quality Partners data on patient experience comes from surveys (with communication, integration, knowledge of patient, access, office staff, and willingness to recommend domains), and its data on screening rates for breast, colorectal, and cervical cancers is derived from chart abstraction. Relative to comparison practices, the communication score in intervention practices increased by 1.47 percentage points on a 100-point scale ( P = .02) between pre and post periods. We did not detect immediate improvements in other measures of patient experience of care and recommended cancer screening rates. Communication may be the first dimension of patient experience that improves following establishment of team-based primary care, and changing care processes may require more time or attention in the transition to team-based care. Our findings also suggest a need to better understand the variation in implementation factors that facilitate some practices’ successful transitions to team-based care, and to use teams effectively to improve cancer screening processes.

Maintaining the essential functions of mitochondria requires mechanisms to recognize and remove misfolded proteins. However, quality control (QC) pathways for misfolded mitochondrial proteins remain poorly defined. Here, we establish temperature-sensitive (ts-) peripheral mitochondrial outer membrane (MOM) proteins as novel model QC substrates in Saccharomyces cerevisiae. The ts- proteins sen2-1HAts and sam35-2HAts are degraded from the MOM by the ubiquitin-proteasome system. Ubiquitination of sen2-1HAts is mediated by the ubiquitin ligase (E3) Ubr1, while sam35-2HAts is ubiquitinated primarily by San1. Mitochondria-associated degradation (MAD) of both substrates requires the SSA family of Hsp70s and the Hsp40 Sis1, providing the first evidence for chaperone involvement in MAD. In addition to a role for the Cdc48-Npl4-Ufd1 AAA-ATPase complex, Doa1 and a mitochondrial pool of the transmembrane Cdc48 adaptor, Ubx2, are implicated in their degradation. This study reveals a unique QC pathway comprised of a combination of cytosolic and mitochondrial factors that distinguish it from other cellular QC pathways.

ObjectiveObservational tuberculosis cohort studies are often limited by a lack of long-term data characterizing survival beyond the initial treatment outcome. Though Cox proportional hazards models are often applied to these data, differential risk of long-term survival, dependent on the initial treatment outcome, can lead to violations of model assumptions. We evaluate the performance of two alternate censoring approaches on reducing bias in treatment effect estimates.DesignWe simulate a typical multidrug-resistant tuberculosis cohort study and use Cox proportional hazards models to assess the relationship of an aggressive treatment regimen with hazard of death. We compare three assumptions regarding censored observations to determine which produces least biased treatment effect estimates: conventional non-informative censoring, an extension of short-term survival informed by literature, and incorporation of predicted long-term vital status.ResultsThe treatment regimen's protective effect on death is consistently underestimated by the conventional censoring method, up to 7.6%. Models using the two alternative censoring techniques produce treatment effect estimates consistently stronger and less biased than the conventional method, underestimating the treatment effect by less than 2.4% across all scenarios.ConclusionsWhen model assumptions are violated, alternative censoring techniques can more accurately estimate associations between treatment and long-term survival. In multidrug-resistant tuberculosis cohort analyses, this bias reduction may yield more accurate and, larger effect estimates. This bias reduction can be achieved through use of standard statistical procedures with a simple re-coding of the censoring indicator.

Environmental DNA (eDNA) can be used for early detection, population estimations, and assessment of potential spread of invasive species, but questions remain about factors that influence eDNA detection results. Efforts are being made to understand how physical, chemical, and biological factors-settling, resuspension, dispersion, eDNA stability/decay-influence eDNA estimations and potentially population abundance. In a series of field and controlled mesocosm experiments, we examined the detection and accumulation of eDNA in sediment and water and the transport of eDNA in a small stream in the Lake Michigan watershed, using the invasive round goby fish (Neogobius melanostomus) as a DNA source. Experiment 1: caged fish (average n = 44) were placed in a stream devoid of round goby; water was collected over 24 hours along 120-m of stream, including a simultaneous sampling event at 7 distances from DNA source; stream monitoring continued for 24 hours after fish were removed. Experiment 2: round goby were placed in laboratory tanks; water and sediment were collected over 14 days and for another 150 days post-fish removal to calculate eDNA shedding and decay rates for water and sediment. For samples from both experiments, DNA was extracted, and qPCR targeted a cytochrome oxidase I gene (COI) fragment specific to round goby. Results indicated that eDNA accumulated and decayed more slowly in sediment than water. In the stream, DNA shedding was markedly lower than calculated in the laboratory, but models indicate eDNA could potentially travel long distances (up to 50 km) under certain circumstances. Collectively, these findings show that the interactive effects of ambient conditions (e.g., eDNA stability and decay, hydrology, settling-resuspension) are important to consider when developing comprehensive models. Results of this study can help resource managers target representative sites downstream of potential invasion sites, thereby maximizing resource use.

The health needs of adolescents and young adults (AYAs) have been neglected in tuberculosis (TB) care, control, and research. AYAs, who are distinct from younger children and older adults, undergo dynamic physical, psychological, emotional, cognitive, and social development. Five domains of adolescent well-being are crucial to a successful transition between childhood and adulthood: (1) Good health; (2) connectedness and contribution to society; (3) safety and a supportive environment; (4) learning, competence, education, skills, and employability; and (5) agency and resilience. This review summarizes the evidence of the impact of TB disease and treatment on these five domains of AYA well-being.

Protein degradation by the ubiquitin-proteasome system is essential to many processes. We sought to assess its involvement in the turnover of mitochondrial proteins in Saccharomyces cerevisiae. We find that deletion of a specific ubiquitin ligase (E3), Psh1p, increases the abundance of a temperature-sensitive mitochondrial protein, mia40-4pHA, when it is expressed from a centromeric plasmid. Deletion of Psh1p unexpectedly elevates the levels of other proteins expressed from centromeric plasmids. Loss of Psh1p does not increase the rate of turnover of mia40-4pHA, affect total protein synthesis, or increase the protein levels of chromosomal genes. Instead, psh1Δ appears to increase the incidence of missegregation of centromeric plasmids relative to their normal 1:1 segregation. After generations of growth with selection for the plasmid, ongoing missegregation would lead to elevated plasmid DNA, mRNA, and protein, all of which we observe in psh1Δ cells. The only known substrate of Psh1p is the centromeric histone H3 variant Cse4p, which is targeted for proteasomal degradation after ubiquitination by Psh1p. However, Cse4p overexpression alone does not phenocopy psh1Δ in increasing plasmid DNA and protein levels. Instead, elevation of Cse4p leads to an apparent increase in 1:0 plasmid segregation events. Further, 2 μm high-copy yeast plasmids also missegregate in psh1Δ, but not when Cse4p alone is overexpressed. These findings demonstrate that Psh1p is required for the faithful inheritance of both centromeric and 2 μm plasmids. Moreover, the effects that loss of Psh1p has on plasmid segregation cannot be accounted for by increased levels of Cse4p.

ABSTRACT The orphan regulator RocA plays a critical role in the colonization and pathogenesis of the obligate human pathogen group A Streptococcus. Despite multiple lines of evidence supporting a role for RocA as an auxiliary regulator of the control of virulence two-component regulatory system CsrRS (or CovRS), the mechanism of action of RocA remains unknown. Using a combination of in vitro and in vivo techniques, we now find that RocA interacts with CsrS in the streptococcal membrane via its N-terminal region, which contains seven transmembrane domains. This interaction is essential for RocA-mediated regulation of CsrRS function. Furthermore, we demonstrate that RocA forms homodimers via its cytoplasmic domain. The serotype-specific RocA truncation in M3 isolates alters this homotypic interaction, resulting in protein aggregation and impairment of RocA-mediated regulation. Taken together, our findings provide insight into the molecular requirements for functional interaction of RocA with CsrS to modulate CsrRS-mediated gene regulation. IMPORTANCE Bacterial two-component regulatory systems, comprising a membrane-bound sensor kinase and cytosolic response regulator, are critical in coordinating the bacterial response to changing environmental conditions. More recently, auxiliary regulators which act to modulate the activity of two-component systems, allowing integration of multiple signals and fine-tuning of bacterial responses, have been identified. RocA is a regulatory protein encoded by all serotypes of the important human pathogen group A Streptococcus. Although RocA is known to exert its regulatory activity via the streptococcal two-component regulatory system CsrRS, the mechanism by which it functions was unknown. Based on new experimental evidence, we propose a model whereby RocA interacts with CsrS in the streptococcal cell membrane to enhance CsrS autokinase activity and subsequent phosphotransfer to the response regulator CsrR, which mediates transcriptional repression of target genes.

Restoration of degraded aquatic habitats is critical to preserve and maintain ecosystem processes and economic viability. Effective restoration requires contaminant sources identification. Microbial communities are increasingly used to characterize fecal contamination sources. The objective was to determine whether nearshore and adjacent beach bacterial contamination originated from the Grand Calumet River, a highly urbanized aquatic ecosystem, and to determine if there were correlations between pathogens/feces associated bacteria in any of the samples to counts of the pathogen indicator species Escherichia coli. Water samples were collected from the river, river mouth, nearshore, and offshore sites along southern Lake Michigan. Comparisons among communities were made using beta diversity distances (weighted and unweighted Unifrac, and Bray Curtis) and Principal Coordinate Analysis of 16S rRNA gene Illumina sequence data that indicated river bacterial communities differed significantly from the river mouth, nearshore lake, and offshore lake samples. These differences were further supported using Source Tracker software that indicated nearshore lake communities differed significantly from river and offshore samples. Among locations, there was separation by sampling date that was associated with environmental factors (e.g., water and air temperature, water turbidity). Although about half the genera (48.1%) were common to all sampling sites, linear discriminant analysis effect size indicated there were several taxa that differed significantly among sites; there were significant positive correlations of feces-associated genera with E. coli most probable numbers. Results collectively highlight that understanding microbial communities, rather than relying solely on select fecal indicators with uncertain origin, are more useful for developing strategies to restore degraded aquatic habitats.

OBJECTIVE:Pakistan has a high pediatric burden of tuberculosis, but few studies describe the treatment experience of children with tuberculosis in Pakistan. We sought to identify risk factors for unsuccessful treatment outcomes in children with drug-susceptible tuberculosis identified in eight hospitals in Karachi, Pakistan. DESIGN:We conducted a retrospective cohort study among children (

ObjectiveTo compare the diagnostic performance of the GenoType MRBDRplus assay with the gold standard phenotypic drug susceptibility testing in the detection of drug resistance among culture isolates obtained from patients in Karachi, Pakistan.DesignMycobacterium tuberculosis isolates were obtained from 96 consecutive tuberculosis patients found to have resistance to isoniazid from two health centers in Karachi (January-November 2017). Isolates were tested for drug resistance against rifampin and isoniazid using the MTBDRplus assay. Results were compared with conventional drug-susceptibility testing and the frequency of specific mutations were reported.ResultsThe MTBDRplus assay had a sensitivity for rifampin resistance of 98.8% (95% CI: 93.4-100) and for isoniazid resistance of 90.6% (95% CI: 83.0-95.6). The MTBDRplus assay showed mutations in rpoB in 81 of the 96 (84.4%) isolates. Of the 87 isolates showing resistance to isoniazid via the MTBDRplus assay, 71 (74.0%) isolates had mutations in the katG gene only, 15 (15.6%) isolates had mutations in the inhA promoter region, and 1 (1.0%) showed mutations in both genes.ConclusionThe GenoType MTBDRplus assay in Pakistan can identify subgroups at high-risk of having isolates with mutations in the katG and/or inhA genes. Understanding the local burden of these mutations have implications for local diagnostic and treatment guidelines.

Background In the United States (U.S.), large price increases for selected generic drugs have elicited public outrage. Recent legislative proposals aim to increase price transparency and identify outlier drug “price spikes.” It is unknown how many and what types of products would be highlighted by such efforts. Methods IQVIA Health Incorporated’s National Sales Perspectives™ provided sales, use and price data for all generic prescription products (unique molecule-manufacturer-formulation combinations) sold in the U.S. We estimated annual prescription price levels and changes between 2013 and 2014. We identify drugs with annual prescription price increases in excess of the medical consumer price index (CPI), and in excess of 15% or 20%, per legislative proposals. We reported annualized inflation-adjusted mean, standard deviation (SD), median, and 95th percentile prescription price increases and percentage of products exceeding the growth in the medical CPI. We fitted logistic regression models to identify characteristics of drugs associated with each category of price increase. Results We analyzed data for 6,182 generic products. The mean inflation-adjusted price increase among all generic products was 38% (SD 1,053%), the median, 2%; the 95th percentile, 135%; and the mean price level, $29.69 (SD $378.44). Approximately half of all products experienced price increases in excess of the growth in the medical CPI; 28% had price increases greater than 15% and 23% had price increases greater than 20%. Drugs exceeding outlier thresholds exhibited lower baseline price levels than the mean price level observed among all generic drugs. The most consistent characteristic predicting whether a product would exceed “price spike” thresholds proposed in legislation is the being supplied by only one manufacturer. Conclusions “Price spikes” among generic drugs in 2014 were more common than newspaper stories and legislative hearings suggest. While the cross-sectional association between an indicator of being sold by only a single manufacturer and the probability of meeting specific price growth thresholds is suggestive of an economically intuitive causal story, future work should delve more deeply into whether decreases in generic competition explain the dramatic price increases that have captured the public’s attention in recent years.

Environmental DNA (eDNA) is revolutionizing biodiversity monitoring, occupancy estimates, and real-time detections of invasive species. In the Great Lakes, the round goby (Neogobius melanostomus), an invasive benthic fish from the Black Sea, has spread to encompass all five lakes and many tributaries, outcompeting or consuming native species; however, estimates of round goby abundance are confounded by behavior and habitat preference, which impact reliable methods for estimating their population. By integrating eDNA into round goby monitoring, improved estimates of biomass may be obtainable. We conducted mesocosm experiments to estimate rates of goby DNA shedding and decay. Further, we compared eDNA with several methods of traditional field sampling to compare its use as an alternative/complementary monitoring method. Environmental DNA decay was comparable to other fish species, and first-order decay was lower at 12°C (k = 0.043) than at 19°C (k = 0.058). Round goby eDNA was routinely detected in known invaded sites of Lake Michigan and its tributaries (range log10 4.8-6.2 CN/L), but not upstream of an artificial fish barrier. Traditional techniques (mark-recapture, seining, trapping) in Lakes Michigan and Huron resulted in fewer, more variable detections than eDNA, but trapping and eDNA were correlated (Pearson R = 0.87). Additional field testing will help correlate round goby abundance with eDNA, providing insight on its role as a prey fish and its impact on food webs.

Ignatzschineria indica is a Gram-negative bacterium that is commonly associated with the larvae of flesh flies. I. indica is difficult to isolate in routine laboratory procedures but has been associated with neglected wounds infested with maggots, fever, elevated white blood count and C-reactive protein, and polymicrobial culture results. Other specific hematological/immunological changes are not known. We present a case of I. indica bacteremia and polymicrobial osteomyelitis resulting from infected decubitus ulcers. The patient improved after treatment with cefepime followed by levofloxacin.

Treatment outcomes for adolescents with multidrug-resistant tuberculosis are rarely reported and, to date, have been poor. Among 90 adolescents from Lima, Peru, 68 (75.6%) achieved cure or completion of treatment. Unsuccessful treatment was less common in the Peru cohort than previously described in the literature.

Human task performance is affected by exposure to physiological and psychological stress. The ability to measure the physiological response to stressors and correlate that to task performance could be used to identify resilient individuals or those at risk for stress-related performance decrements. Accomplishing this prior to performance under severe stress or the development of clinical stress disorders could facilitate focused preparation such as tailoring training to individual needs. Here we measure the effects of stress on physiological response and performance through behavior, physiological sensors, and subjective ratings, and identify which individuals are at risk for stress-related performance decrements. Participants performed military-relevant training tasks under stress in a virtual environment, with autonomic and hypothalamic-pituitary-adrenal axis (HPA) reactivity analyzed. Self-reported stress, as well as physiological indices of stress, increased in the group pre-exposed to socioevaluative stress. Stress response was effectively captured via electrodermal and cardiovascular measures of heart rate and skin conductance level. A resilience classification algorithm was developed based upon physiological reactivity, which correlated with baseline unstressed physiological and self-reported stress values. Outliers were identified in the experimental group that had a significant mismatch between self-reported stress and salivary cortisol. Baseline stress measurements were predictive of individual resilience to stress, including the impact stress had on physiological reactivity and performance. Such an approach may have utility in identifying individuals at risk for problems performing under severe stress. Continuing work has focused on adapting this method for military personnel, and assessing the utility of various coping and decision-making strategies on performance and physiological stress.

Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators, have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson’s disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (< 5 kb). In utero electroporation offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5-14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal activity in the rat brain.