Clinical and economic impacts of a modified-observational screening approach to well-appearing infants born to mothers with chorioamnionitis.

OBJECTIVE: Term infants born to mothers with chorioamnionitis are at risk for early-onset sepsis (EOS). We aimed to measure the impact of changing from a categorical to a modified-observational EOS screening approach on NICU admission, antibiotic utilization, and hospitalization costs. STUDY DESIGN: Single-center retrospective pre-post cohort study of full-term infants born to mothers with chorioamnionitis. Primary outcomes included NICU admission, antibiotic utilization, and hospitalization costs. Outcomes were adjusted for demographic variables. Budget-impact analysis was performed using bootstrapping with replication. RESULTS: 380 term infants were included (197 categorical; 183 modified-observational). There was a significant decrease in NICU admission and antibiotic utilization (p < 0.05) in the modified-observational cohort but no significant difference in per-patient total hospitalization costs. Budget-impact analysis suggested a high probability of cost savings. CONCLUSION: A modified-observational approach to evaluating term infants of mothers with chorioamnionitis can reduce NICU admission and unnecessary antibiotic therapy, and may lead to cost-savings.

Current Trends in Invasive Closure of Patent Ductus Arteriosus in Very Low Birth Weight Infants in United States Children's Hospitals, 2016-2021.

OBJECTIVE: To describe the current practices in invasive patent ductus arteriosus (PDA) closure (surgical ligation or transcatheter occlusion) in very low birth weight (VLBW) infants and changes in patient characteristics and outcomes from 2016 to 2021 among US children's hospitals. STUDY DESIGN: We evaluated a retrospective cohort of VLBW infants (birth weight 400-1499 g and gestational age 22-31 weeks) who had invasive PDA closure within 6 months of age from 2016 to 2021 in children's hospitals in the Pediatric Health Information System. Changes in patient characteristics and outcomes over time were evaluated using generalized linear models and generalized linear mixed models. RESULTS: 2418 VLBW infants (1182 surgical ligation; 1236 transcatheter occlusion) from 42 hospitals were included. The proportion of infants receiving transcatheter occlusion increased from 17.2% in 2016 to 84.4% in 2021 (P < .001). In 2021, 28/42 (67%) hospitals had performed transcatheter occlusion in > 80% of their VLBW infants needing invasive PDA closure, compared with only 2/42 (5%) in 2016. Although median postmenstrual age (PMA) at PDA closure did not change for the overall cohort, PMA at transcatheter occlusion decreased from 38 weeks in 2016 to 31 weeks by 2020, P < .001. Among those infants not intubated prior to PDA closure, extubation within 3 days postprocedure increased over time (yearly adjusted odds ratios of 1.26 [1.08-1.48]). Length of stay and mortality did not change over time. CONCLUSION: We report rapid adoption of transcatheter occlusion for PDA among VLBW infants in US children's hospitals over time. Transcatheter occlusions were performed at younger PMA over time.

Hospital variation in neonatal echocardiography among very preterm infants at US children's hospitals.

OBJECTIVE: Estimate hospital variation in echocardiography (echo) among very preterm infants. STUDY DESIGN: Retrospective cohort study of very preterm (<32 weeks) infants discharged between 2012 and 2019 from US children's hospitals. Echo exposure was identified using daily billing, and hospital variation was estimated after adjustment for illness severity. Variation in very early echo use (<3 days of life) was compared to exposure to treatment of a patent ductus arteriosus (PDA), and other practice patterns. RESULTS: 27,498 subjects across 39 children's hospitals were included. Very early echo use had the greatest hospital variation (3-34%). Increasing very early echo use was not associated with PDA treatment (p = 0.93), but was associated with nitric oxide (p < 0.01) and vasoactive medications (p < 0.01). CONCLUSIONS: Hospital variation in echo use among preterm infants was greatest in the first few days of life and was associated with increasing nitric oxide and vasoactive medication use. The impact of this variation on clinical outcomes is uncertain and warrants further investigation.

Two-Tiered Selection and Screening Strategy to Increase Functional Enzyme Production in E. coli.

Development of recombinant enzymes as industrial biocatalysts or metabolic pathway elements requires soluble expression of active protein. Here we present a two-step strategy, combining a directed evolution selection with an enzyme activity screen, to increase the soluble production of enzymes in the cytoplasm of E. coli. The directed evolution component relies on the innate quality control of the twin-arginine translocation pathway coupled with antibiotic selection to isolate point mutations that promote intracellular solubility. A secondary screen is applied to ensure the solubility enhancement has not compromised enzyme activity. This strategy has been successfully applied to increase the soluble production of a fungal endocellulase by 30-fold in E. coli without change in enzyme specific activity through two rounds of directed evolution.

Economic considerations at the threshold of viability.

Neonatal intensive care for infants born at 22-24 weeks has become more prevalent in the past three decades, but outcomes remain highly variable between centers, in part due to different approaches in management. With this increased frequency of intervention, there has been concern for a concurrent increase in costs of care for survivors. This article reviews the direct medical, direct non-medical, and indirect costs of care for periviable infants and their families, as well as the current limitations of published data. In addition, we highlight the cost-effectiveness of neonatal intensive care and various therapies offered to extremely preterm infants, while also considering the ethical dilemmas inherently tied to periviable decision-making. Strategies to improve the gaps in knowledge on the economic impact of the smallest infants are discussed.

Prioritization framework for improving the value of care for very low birth weight and very preterm infants.

OBJECTIVE: Create a prioritization framework for value-based improvement in neonatal care. STUDY DESIGN: A retrospective cohort study of very low birth weight (<1500 g) and/or very preterm (<32 weeks) infants discharged between 2012 and 2019 using the Pediatric Health Information System Database. Resource use was compared across hospitals and adjusted for patient-level differences. A prioritization score was created combining cost, patient exposure, and inter-hospital variability to rank resource categories. RESULTS: Resource categories with the greatest cost, patient exposure, and inter-hospital variability were parenteral nutrition, hematology (lab testing), and anticoagulation (for central venous access and therapy), respectively. Based on our prioritization score, parenteral nutrition was identified as the highest priority overall. CONCLUSIONS: We report the development of a prioritization score for potential value-based improvement in neonatal care. Our findings suggest that parenteral nutrition, central venous access, and high-volume laboratory and imaging modalities should be priorities for future comparative effectiveness and quality improvement efforts.

The financial burden on families of infants requiring neonatal intensive care.

While the high costs of neonatal intensive care have been a topic of increasing study, the financial impact on families have been less frequently reported or summarized. We conducted a systematic review of the literature using Pubmed/Medline and EMBASE (1990-2020) for studies reporting estimates of out-of-pocket costs or qualitative estimates of financial burden on families during a neonatal intensive care unit stay or after discharge. 44 studies met inclusion criteria, with 25 studies providing cost estimates. Cost estimates primarily focused on direct non-medical out-of-pocket costs or loss of productivity, and there was a paucity of cost estimates for insurance cost-sharing. Available estimates suggest these costs are significant to families, cause significant stress, and may impact care received by patients. More high-quality studies estimating the entirety of out-of-pocket costs are needed, and particular attention should be paid to how these costs directly impact the care of our high-risk population.

Cost of clinician-driven tests and treatments in very low birth weight and/or very preterm infants.

OBJECTIVE: To rank clinician-driven tests and treatments (CTTs) by their total cost during the birth hospitalization for preterm infants. STUDY DESIGN: Retrospective cohort of very low birth weight (<1500 g) and/or very preterm (<32 weeks) subjects admitted to US children's hospital Neonatal Intensive Care Units (2012-2018). CTTs were defined as pharmaceutical, laboratory and imaging services and ranked by total cost. RESULTS: 24,099 infants from 51 hospitals were included. Parenteral nutrition ($85M, 32% of pharmacy costs), blood gas analysis ($34M, 29% of laboratory costs), and chest radiographs ($18M, 31% of imaging costs) were the costliest CTTs overall. More than half of CTT-related costs occurred during 10% of hospital days. CONCLUSIONS: The majority of CTT-related costs were from commonly used tests and treatments. Targeted efforts to improve value in neonatal care may benefit most from focusing on reducing unnecessary utilization of common tests and treatments, rather than infrequently used ones.

Optimizing the Use of Human Milk Cream Supplement in Very Preterm Infants: Growth and Cost Outcomes.

BACKGROUND: An exclusive human milk-based diet has been shown to decrease necrotizing enterocolitis and improve outcomes for infants ≤1250 g birth weight. Studies have shown that infants who received an exclusive human milk diet with a donor-human milk-derived cream supplement (cream) had improved weight and length velocity when the cream was added to mother's own milk or donor-human milk when energy was <20 kcal/oz using a human milk analyzer. Our objective was to compare growth and cost outcomes of infants ≤1250 g birth weight fed with an exclusive human milk diet, with and without human milk cream, without the use of a human milk analyzer. METHODS: Two cohorts of human milk-fed premature infants were compared from birth to 34 weeks postmenstrual age. Group 1 (2010-2011) received a donor-human milk fortifier, whereas Group 2 (2015-2016) received donor-human milk fortifier plus the commercial cream supplement, if weight gain was <15 g/kg/d. RESULTS: There was no difference in growth between the 2 groups for weight (P = 0.32) or head circumference (P = 0.90). Length velocity was greater for Group 1 (P = 0.03). The mean dose of donor-human milk fortifier was lower in Group 2 (P < 0.001). Group 2 saved an average of $2318 per patient on the cost of human milk products (P < 0.01). CONCLUSIONS: Infants receiving a human milk diet with cream supplementation for growth faltering achieve appropriate growth in a cost-effective feeding strategy.

Mask versus Prongs for Nasal Continuous Positive Airway Pressure in Preterm Infants: A Systematic Review and Meta-Analysis.

Nasal continuous positive airway pressure (NCPAP) is an effective method of respiratory support for preterm infants. Nasal masks and binasal prongs are two interfaces available to deliver NCPAP, and it is unclear if one is superior to the other. We conducted a systematic review and meta-analysis, using the methodology recommended by the Cochrane Collaboration, to compare the efficacy and safety of nasal masks versus binasal prongs to deliver NCPAP in preterm infants <37 weeks of gestation. Ovid MEDLINE, Embase, Scopus, the Cochrane database, and PubMed were searched in February 2019. Seven trials met the inclusion criteria. Among preterm infants requiring NCPAP, the use of a nasal mask, compared to nasal prongs, decreased the rate of NCPAP failure within 72 h (RR 0.72, 95% CI 0.53-0.97; number needed to treat for an additional beneficial outcome [NNTB] 12.5, 95% CI 7.1-100; 5 trials, 576 participants; low-certainty evidence) and the incidence of nasal injury (RR 0.71, 95% CI 0.59-0.85; NNTB 8.3, 95% CI 5.6-16.7; 6 trials, 665 participants; low-certainty evidence). In a subgroup of preterm infants requiring NCPAP after resuscitation at birth, the use of a nasal mask decreased the incidence of moderate-to-severe bronchopulmonary dysplasia (RR 0.47, 95% CI 0.23-0.95; NNTB 16.7, 95% CI 9.1-100; 4 trials, 395 participants; very-low-certainty evidence) and the need for subsequent surfactant administration (RR 0.78, 95% CI 0.64-0.96; NNTB 8.33, 95% CI 4.54-33.33; 4 trials, 395 participants; low-certainty evidence). The use of nasal masks for preterm infants requiring NCPAP was associated with a reduction in NCPAP failure, need for surfactant administration, and moderate-to-severe bronchopulmonary dysplasia (low- to very-low-certainty evidence). Given the potential clinical benefit and minimal risk associated with a change in patient interface, nasal masks should be considered the preferred interface for NCPAP delivery in preterm infants.

Pediatric Training Faculty and Resident Perceptions on Teaching High-Value, Cost-Conscious Care: A Multi-Institutional Study.

BACKGROUND AND OBJECTIVES: High-value, cost-conscious care (HVCCC) is care that promotes optimal patient outcomes while reducing unnecessary costs. Teaching to promote HVCCC is essential, yet little research has assessed the dual perspectives of residents and faculty on this topic. Our aim was to investigate pediatric resident and faculty perspectives of HVCCC training and role modeling to more effectively promote curriculum and faculty development on this subject. METHODS: Pediatric residents and teaching faculty in 2 academic medical centers were surveyed during the 2015-2016 academic year. Questions addressed comfort with HVCCC, current teaching practices, barriers to teaching HVCC, and desired curriculum. Descriptive statistics were used to summarize data, and Fisher's exact or χ2 tests were used to assess for associations between responses. Institutional review board approval was obtained at both participating institutions. RESULTS: We received responses from 51% of faculty (128 of 249) and 60% of residents (73 of 123). Most faculty and residents agreed that HVCCC training is important, but only 26% of residents (18 of 69) felt comfortable practicing HVCCC. Faculty and residents identified lack of training or knowledge (50%, 61 of 121 and 53%, 37 of 70, respectively) and lack of hospital support (73%, 88 of 121 and 69%, 47 of 68, respectively) as the largest barriers. Of residents, >85% (60 of 69) reported a lack of attending physician role modeling. Most faculty (83%, 102 of 123) desired faculty development. CONCLUSIONS: Residents and faculty agree that HVCCC is important and that training institutions have a responsibility to address it. However, most residents were not comfortable with HVCCC. Faculty development in HVCCC as well as hospital support for access to pricing data will be key for programs to develop effective resident training in this area.

Stable heterologous expression of biologically active terpenoids in green plant cells.

Plants biosynthesize a great diversity of biologically active small molecules of interest for fragrances, flavors, and pharmaceuticals. Among specialized metabolites, terpenoids represent the greatest molecular diversity. Many terpenoids are very complex, and total chemical synthesis often requires many steps and difficult chemical reactions, resulting in a low final yield or incorrect stereochemistry. Several drug candidates with terpene skeletons are difficult to obtain by chemical synthesis due to their large number of chiral centers. Thus, biological production remains the preferred method for industrial production for many of these compounds. However, because these chemicals are often found in low abundance in the native plant, or are produced in plants which are difficult to cultivate, there is great interest in engineering increased production or expression of the biosynthetic pathways in heterologous hosts. Although there are many examples of successful engineering of microbes such as yeast or bacteria to produce these compounds, this often requires extensive changes to the host organism's metabolism. Optimization of plant gene expression, post-translational protein modifications, subcellular localization, and other factors often present challenges. To address the future demand for natural products used as drugs, new platforms are being established that are better suited for heterologous production of plant metabolites. Specifically, direct metabolic engineering of plants can provide effective heterologous expression for production of valuable plant-derived natural products. In this review, our primary focus is on small terpenoids and we discuss the benefits of plant expression platforms and provide several successful examples of stable production of small terpenoids in plants.

Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.

Heterologous expression of many proteins in bacteria, yeasts, and plants is often limited by low titers of functional protein. To address this problem, we have created a two-tiered directed evolution strategy in Escherichia coli that enables optimization of protein production while maintaining high biological activity. The first tier involves a genetic selection for intracellular protein stability that is based on the folding quality control mechanism inherent to the twin-arginine translocation pathway, while the second is a semi-high-throughput screen for protein function. To demonstrate the utility of this strategy, we isolated variants of the endoglucanase Cel5A, from the plant-pathogenic fungus Fusarium graminearum, whose production was increased by as much as 30-fold over the parental enzyme. This gain in production was attributed to just two amino acid substitutions, and it was isolated after two iterations through the two-tiered approach. There was no significant tradeoff in activity on soluble or insoluble cellulose substrates. Importantly, by combining the folding filter afforded by the twin-arginine translocation quality control mechanism with a function-based screen, we show enrichment for variants with increased protein abundance in a manner that does not compromise catalytic activity, providing a highly soluble parent for engineering of improved or new function.

Plant pathogens as a source of diverse enzymes for lignocellulose digestion.

The plant cell wall is a major barrier that many plant pathogens must surmount for successful invasion of their plant hosts. Full genome sequencing of a number of plant pathogens has revealed often large, complex, and redundant enzyme systems for degradation of plant cell walls. Recent surveys have noted that plant pathogenic fungi are highly competent producers of lignocellulolytic enzymes, and their enzyme activity patterns reflect host specificity. We propose that plant pathogens may contribute to biofuel production as diverse sources of accessory enzymes for more efficient conversion of lignocellulose into fermentable sugars.

Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi.

BACKGROUND: The discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for the production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly cellulolytic and is a major industrial microbial source for commercial cellulases, xylanases and other cell wall degrading enzymes. However, enzyme-prospecting research continues to identify opportunities to enhance the activity of T. reesei enzyme preparations by supplementing with enzymatic diversity from other microbes. The goal of this study was to evaluate the enzymatic potential of a broad range of plant pathogenic and non-pathogenic fungi for their ability to degrade plant biomass and isolated polysaccharides. RESULTS: Large-scale screening identified a range of hydrolytic activities among 348 unique isolates representing 156 species of plant pathogenic and non-pathogenic fungi. Hierarchical clustering was used to identify groups of species with similar hydrolytic profiles. Among moderately and highly active species, plant pathogenic species were found to be more active than non-pathogens on six of eight substrates tested, with no significant difference seen on the other two substrates. Among the pathogenic fungi, greater hydrolysis was seen when they were tested on biomass and hemicellulose derived from their host plants (commelinoid monocot or dicot). Although T. reesei has a hydrolytic profile that is highly active on cellulose and pretreated biomass, it was less active than some natural isolates of fungi when tested on xylans and untreated biomass. CONCLUSIONS: Several highly active isolates of plant pathogenic fungi were identified, particularly when tested on xylans and untreated biomass. There were statistically significant preferences for biomass type reflecting the monocot or dicot host preference of the pathogen tested. These highly active fungi are promising targets for identification and characterization of novel cell wall degrading enzymes for industrial applications.

Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism.

Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.

An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts.

Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting, a standardized microplate assay was developed for rapid analysis of polysaccharide hydrolysis by fungal extracts, incorporating biomass substrates. Fungi were grown for 10 days on cellulose- or switchgrass-containing media to produce enzyme extracts for analysis. Reducing sugar released from filter paper, Avicel, corn stalk, switchgrass, carboxymethylcellulose, and arabinoxylan was quantified using a miniaturized colorimetric assay based on 3,5-dinitrosalicylic acid. Significant interactions were identified among fungal species, growth media composition, assay substrate, and temperature. Within a small sampling of plant pathogenic fungi, some extracts had crude activities comparable to or greater than T. reesei, particularly when assayed at lower temperatures and on biomass substrates. This microplate assay system should prove useful for high-throughput bioprospecting for new sources of novel enzymes for biofuel production.