Production of native recombinant proteins using a novel split intein affinity technology.

Affinity tags are frequently engineered into recombinant proteins to facilitate purification. Although this technique is powerful, removal of the tag is desired because the tag can interfere with biological activity and can potentially increase the immunogenicity of therapeutic proteins. Tag removal is complex, as it requires adding expensive protease enzymes. To overcome this limitation, split intein based affinity purification systems have been developed in which a CC-intein tag is engineered into a protein of interest for binding to a NC-intein peptide ligand fixed to a chromatographic support. Tag removal in these systems is achieved by creating an active intein-complex during protein capture, which triggers a precise self-cleavage reaction. In this work, we show applications of a new split intein system, Cytiva™ ProteinSelect™. One advantage of the new system is that the NC-intein ligand can be robustly produced and conjugated to large volumes of resin for production of gram scale proteins. SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager in this work were successfully captured on the affinity resin and scaled 10-fold. Another advantage of this system is the ability to sanitize the resin with sodium hydroxide without loosing the 10-20 g/L binding capacity. Binding studies with IL-1b and IFNAR-1 ECD showed that the resin can be regenerated and sanitized for up to 50 cycles without loosing binding capacity. Additionally, after several cycles of sanitization, binding capacity was retained for the SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager. As with other split intein systems, optimization was needed to achieve ideal expression and recovery. The N-terminal amino acid sequence of the protein of interest required engineering to enable the cleavage reaction. Additionally, ensuring the stability of the CC-intein tag was important to prevent premature cleavage or truncation. Controlling the hold time of the expression product and the prevention of protease activity prior to purification was needed. These results demonstrate the feasibility of the Cytiva™ ProteinSelect™ system to be used in academic and industrial research and development laboratories for the purification of novel proteins expressed in either bacterial or mammalian systems.

Interim safety and immunogenicity of COVID-19 omicron BA.1 variant-containing vaccine in children in the USA: an open-label non-randomised phase 3 trial.

BACKGROUND: Variant-containing mRNA vaccines for COVID-19 to broaden protection against SARS-CoV-2 variants are recommended based on findings in adults. We report interim safety and immunogenicity of an omicron BA.1 variant-containing (mRNA-1273.214) primary vaccination series and booster dose in paediatric populations. METHODS: This open-label, two-part, non-randomised phase 3 trial enrolled participants aged 6 months to 5 years at 24 US study sites. Eligible participants were generally healthy or had stable chronic conditions, without known SARS-CoV-2 infection in the previous 90 days. Individuals who were acutely ill or febrile 1 day before or at the screening visit or those who previously received other COVID-19 vaccines (except mRNA-1273 for part 2) were excluded. In part 1, SARS-CoV-2-vaccine-naive participants received two-dose mRNA-1273.214 (25 µg; omicron BA.1 and ancestral Wuhan-Hu-1 mRNA) primary series. In part 2, participants who previously completed the two-dose mRNA-1273 (25 µg) primary series in KidCOVE (NCT04796896) received a mRNA-1273.214 (10 µg) booster dose. Primary study outcomes were safety and reactogenicity of the mRNA-1273.214 primary series (part 1) or booster dose (part 2) as well as the inferred effectiveness of mRNA-1273.214 based on immune responses against ancestral SARS-CoV-2 (D614G) and omicron BA.1 variant at 28 days post-primary series (part 1) or post-booster dose (part 2). The safety set included participants who received at least one dose of the study vaccine; the immunogenicity set included those who provided immunogenicity samples. Interim safety and immunogenicity are summarised in this analysis as of the data cutoff date (Dec 5, 2022). This trial is registered with ClinicalTrials.gov, NCT05436834. FINDINGS: Between June 21, 2022, and Dec 5, 2022, 179 participants received one or more doses of mRNA-1273.214 primary series (part 1) and 539 received a mRNA-1273.214 booster dose (part 2). The safety profile within 28 days after either dose of the mRNA-1273.214 primary series and the booster dose was consistent with that of the mRNA-1273 primary series in this age group, with no new safety concerns or vaccine-related serious adverse events observed. At 28 days after primary series dose 2 and the booster dose, both mRNA-1273.214 primary series (day 57, including all participants with or without evidence of prior SARS-CoV-2 infection at baseline) and booster (day 29, including participants without evidence of prior SARS-CoV-2 infection at baseline) elicited responses that were superior against omicron-BA.1 (geometric mean ratio part 1: 25·4 [95% CI 20·1-32·1] and part 2: 12·5 [11·0-14·3]) and non-inferior against D614G (part 1: 0·8 [0·7-1·0] and part 2: 3·1 [2·8-3·5]), compared with neutralising antibody responses induced by the mRNA-1273 primary series (in a historical comparator group). INTERPRETATION: mRNA-1273.214 was immunogenic against BA.1 and D614G in children aged 6 months to 5 years, with a comparable safety profile to mRNA-1273, when given as a two-dose primary series or a booster dose. These results are aligned with the US Centers for Disease Control and Prevention recommendations for the use of variant-containing vaccines for continued protection against the emerging variants of SARS-CoV-2. FUNDING: Moderna.

Single-cell transcriptomics identifies prothymosin α restriction of HIV-1 in vivo.

Host restriction factors play key roles in innate antiviral defense, but it remains poorly understood which of them restricts HIV-1 in vivo. Here, we used single-cell transcriptomic analysis to identify host factors associated with HIV-1 control during acute infection by correlating host gene expression with viral RNA abundance within individual cells. Wide sequencing of cells from one participant with the highest plasma viral load revealed that intracellular viral RNA transcription correlates inversely with expression of the gene PTMA, which encodes prothymosin α. This association was genome-wide significant (Padjusted < 0.05) and was validated in 28 additional participants from Thailand and the Americas with HIV-1 CRF01_AE and subtype B infections, respectively. Overexpression of prothymosin α in vitro confirmed that this cellular factor inhibits HIV-1 transcription and infectious virus production. Our results identify prothymosin α as a host factor that restricts HIV-1 infection in vivo, which has implications for viral transmission and cure strategies.

HLA-B∗46 associates with rapid HIV disease progression in Asian cohorts and prominent differences in NK cell phenotype.

Human leukocyte antigen (HLA) alleles have been linked to HIV disease progression and attributed to differences in cytotoxic T lymphocyte (CTL) epitope representation. These findings are largely based on treatment-naive individuals of European and African ancestry. We assessed HLA associations with HIV-1 outcomes in 1,318 individuals from Thailand and found HLA-B∗46:01 (B∗46) associated with accelerated disease in three independent cohorts. B∗46 had no detectable effect on HIV-specific T cell responses, but this allele is unusual in containing an HLA-C epitope that binds inhibitory receptors on natural killer (NK) cells. Unbiased transcriptomic screens showed increased NK cell activation in people with HIV, without B∗46, and simultaneous single-cell profiling of surface proteins and transcriptomes revealed a NK cell subset primed for increased responses in the absence of B∗46. These findings support a role for NK cells in HIV pathogenesis, revealed by the unique properties of the B∗46 allele common only in Asia.

Genetic variation that determines TAPBP expression levels associates with the course of malaria in an HLA allotype-dependent manner.

HLA class I (HLA-I) allotypes vary widely in their dependence on tapasin (TAPBP), an integral component of the peptide-loading complex, to present peptides on the cell surface. We identified two single-nucleotide polymorphisms that regulate TAPBP messenger RNA (mRNA) expression in Africans, rs111686073 (G/C) and rs59097151 (A/G), located in an AP-2α transcription factor binding site and a microRNA (miR)-4486 binding site, respectively. rs111686073G and rs59097151A induced significantly higher TAPBP mRNA expression relative to the alternative alleles due to higher affinity for AP-2α and abrogation of miR-4486 binding, respectively. These variants associated with lower Plasmodium falciparum parasite prevalence and lower incidence of clinical malaria specifically among individuals carrying tapasin-dependent HLA-I allotypes, presumably by augmenting peptide loading, whereas tapasin-independent allotypes associated with relative protection, regardless of imputed TAPBP mRNA expression levels. Thus, an attenuated course of malaria may occur through enhanced breadth and/or magnitude of antigen presentation, an important consideration when evaluating vaccine efficacy.

Evaluation of mRNA-1273 Covid-19 Vaccine in Children 6 to 11 Years of Age.

BACKGROUND: Vaccination of children to prevent coronavirus disease 2019 (Covid-19) is an urgent public health need. The safety, immunogenicity, and efficacy of the mRNA-1273 vaccine in children 6 to 11 years of age are unknown. METHODS: Part 1 of this ongoing phase 2-3 trial was open label for dose selection; part 2 was an observer-blinded, placebo-controlled expansion evaluation of the selected dose. In part 2, we randomly assigned children (6 to 11 years of age) in a 3:1 ratio to receive two injections of mRNA-1273 (50 µg each) or placebo, administered 28 days apart. The primary objectives were evaluation of the safety of the vaccine in children and the noninferiority of the immune response in these children to that in young adults (18 to 25 years of age) in a related phase 3 trial. Secondary objectives included determination of the incidences of confirmed Covid-19 and severe acute respiratory syndrome coronavirus 2 infection, regardless of symptoms. Interim analysis results are reported. RESULTS: In part 1 of the trial, 751 children received 50-µg or 100-µg injections of the mRNA-1273 vaccine, and on the basis of safety and immunogenicity results, the 50-µg dose level was selected for part 2. In part 2 of the trial, 4016 children were randomly assigned to receive two injections of mRNA-1273 (50 µg each) or placebo and were followed for a median of 82 days (interquartile range, 14 to 94) after the first injection. This dose level was associated with mainly low-grade, transient adverse events, most commonly injection-site pain, headache, and fatigue. No vaccine-related serious adverse events, multisystem inflammatory syndrome in children, myocarditis, or pericarditis were reported as of the data-cutoff date. One month after the second injection (day 57), the neutralizing antibody titer in children who received mRNA-1273 at a 50-µg level was 1610 (95% confidence interval [CI], 1457 to 1780), as compared with 1300 (95% CI, 1171 to 1443) at the 100-µg level in young adults, with serologic responses in at least 99.0% of the participants in both age groups, findings that met the prespecified noninferiority success criterion. Estimated vaccine efficacy was 88.0% (95% CI, 70.0 to 95.8) against Covid-19 occurring 14 days or more after the first injection, at a time when B.1.617.2 (delta) was the dominant circulating variant. CONCLUSIONS: Two 50-µg doses of the mRNA-1273 vaccine were found to be safe and effective in inducing immune responses and preventing Covid-19 in children 6 to 11 years of age; these responses were noninferior to those in young adults. (Funded by the Biomedical Advanced Research and Development Authority and the National Institute of Allergy and Infectious Diseases; KidCOVE ClinicalTrials.gov number, NCT04796896.).

Infection preventionist staffing levels and rates of 10 types of healthcare-associated infections: A 9-year ambidirectional observation.

OBJECTIVE: To quantitatively evaluate relationships between infection preventionists (IPs) staffing levels, nursing hours, and rates of 10 types of healthcare-associated infections (HAIs). DESIGN AND SETTING: An ambidirectional observation in a 528-bed teaching hospital. PATIENTS: All inpatients from July 1, 2012, to February 1, 2021. METHODS: Standardized US National Health Safety Network (NHSN) definitions were used for HAIs. Staffing levels were measured in full-time equivalents (FTE) for IPs and total monthly hours worked for nurses. A time-trend analysis using control charts, t tests, Poisson tests, and regression analysis was performed using Minitab and R computing programs on rates and standardized infection ratios (SIRs) of 10 types of HAIs. An additional analysis was performed on 3 stratifications: critically low (2-3 FTE), below recommended IP levels (4-6 FTE), and at recommended IP levels (7-8 FTE). RESULTS: The observation covered 1.6 million patient days of surveillance. IP staffing levels fluctuated from ≤2 IP FTE (critically low) to 7-8 IP FTE (recommended levels). Periods of highest catheter-associated urinary tract infection SIRs, hospital-onset Clostridioides difficile and carbapenem-resistant Enterobacteriaceae infection rates, along with 4 of 5 types of surgical site SIRs coincided with the periods of lowest IP staffing levels and the absence of certified IPs and a healthcare epidemiologist. Central-line-associated bloodstream infections increased amid lower nursing levels despite the increased presence of an IP and a hospital epidemiologist. CONCLUSIONS: Of 10 HAIs, 8 had highest incidences during periods of lowest IP staffing and experience. Some HAI rates varied inversely with levels of IP staffing and experience and others appeared to be more influenced by nursing levels or other confounders.

Lubiprostone for Pediatric Functional Constipation: Randomized, Controlled, Double-Blind Study With Long-term Extension.

BACKGROUND & AIMS: Pediatric functional constipation (PFC) is a common problem in children that causes distress and presents treatment challenges to health care professionals. We conducted a randomized, placebo-controlled trial (study 1) in patients with PFC (6-17 years of age) to evaluate the efficacy and safety of lubiprostone, followed by an open-label extension for those who completed the placebo-controlled phase (study 2). METHODS: Study 1 (NCT02042183) was a phase 3, multicenter, randomized, double-blind, placebo-controlled, 12-week study evaluating the efficacy and safety of lubiprostone 12 µg twice daily (BID) and 24 µg BID. Study 2 (NCT02138136) was a phase 3, long-term, open-label extension of study 1. In both studies, lubiprostone doses were based on patients' weight. Efficacy was assessed solely based on study 1, with a primary endpoint of overall spontaneous bowel movement (SBM) response (increase of ≥1 SBM/wk vs baseline and ≥3 SBMs/wk for ≥9 weeks, including 3 of the final 4 weeks). RESULTS: 606 patients were randomized to treatment (placebo: n = 202; lubiprostone: n = 404) in study 1. No statistically significant difference in overall SBM response rate was observed between the lubiprostone and placebo groups (18.5% vs 14.4%; P = .2245). Both the 12-µg BID and 24-µg BID doses of lubiprostone were well tolerated in the double-blind and extension phases, with a safety profile consistent with that seen in adult studies. CONCLUSIONS: Lubiprostone did not demonstrate statistically significant effectiveness over placebo in children and adolescents with PFC but did demonstrate a safety profile similar to that in adults. (ClinicalTrials.gov: Number: NCT02042183; Number: NCT02138136).

Immunogenicity and safety of different schedules of the meningococcal ABCWY vaccine, with assessment of long-term antibody persistence and booster responses - results from two phase 2b randomized trials in adolescents.

The meningococcal serogroup B (MenB) protein vaccine, 4CMenB, combined with MenA, MenC, MenW and MenY polysaccharide-protein conjugates for a pentavalent MenABCWY vaccine, can potentially protect against most causative agents of invasive meningococcal disease worldwide. Two phase 2b, randomized, multicenter studies were conducted (NCT02212457, NCT02946385) to assess the immunogenicity and safety of the MenABCWY vaccine as well as antibody persistence and response to a booster dose 2 years after the last vaccination, compared to 4CMenB vaccination. Participants (10 - 18 years), randomized (3:3:2:2:2:2), received the 4-component 4CMenB vaccine according to a 0-2 month (M) schedule or MenABCWY according to a 0-2, 0-6, 0-2-6, 0-1, or 0-11 M schedule. All participants received 5 injections (at M0, M1, M2, M6 and M12) with either the study vaccines or placebo/hepatitis A vaccine. Follow-on participants (4CMenB-0-2, MenABCWY-0-2, MenABCWY-0-6 and MenABCWY-0-2-6 groups) received one dose of either 4CMenB (4CMenB-0-2 group) or MenABCWY and newly enrolled, age-matched, meningococcal vaccine-naïve adolescents (randomized 1:1) received 2 doses (0-2 M) of either 4CMenB or MenABCWY. MenABCWY vaccination was immunogenic against MenB test strains. Non-inferiority for all 4 components of the 4CMenB vaccine could not be demonstrated for the 0-2 M schedule. Antibodies persisted up to 2 years post-MenABCWY vaccination and a booster dose induced an anamnestic response as higher titers were observed in follow-on participants compared to the first-dose response in vaccine-naïve participants. MenABCWY had a clinically-acceptable safety profile, not different from that of 4CMenB.

Safety of Lubiprostone in Pediatric Patients With Functional Constipation: A Nonrandomized, Open-Label Trial.

OBJECTIVES: Pediatric functional constipation (PFC) affects up to 30% of children. Current treatments often do not sustain symptomatic relief. Lubiprostone is a locally acting chloride channel activator that promotes fluid secretion into the small bowel without affecting serum electrolyte concentrations. We assessed the safety/tolerability of oral lubiprostone as treatment for PFC in a 24-week study. METHODS: This phase 3 open-label safety trial conducted from April-November 2016 at 13 US sites included patients (ages 6-17 years) diagnosed with PFC (Rome III criteria). Patients <50 and ≥50 kg received lubiprostone 12 or 24 mcg twice daily, respectively, for 24 weeks. Safety endpoints included incidence of treatment-emergent adverse events (TEAEs) and changes from baseline in clinical laboratory parameters and vital signs. RESULTS: Overall, 87 patients receiving lubiprostone, 64.3% (36/56) in the 12-mcg group and 54.8% (17/31) in the 24-mcg group, completed the study. Of 12 TEAEs leading to discontinuation, only upper abdominal pain occurred in >1 patient. TEAEs were mostly mild in intensity, with gastrointestinal disorders (diarrhea, vomiting) most frequently reported. No safety concerns were found in vital signs, abbreviated physical examinations, and laboratory tests. Subgroup analyses assessed an impact of age, sex, and race categories on TEAEs and treatment-related adverse events. Mean investigators' assessments of treatment effectiveness (scale of 0-4) for lubiprostone 12- and 24-mcg groups, respectively, were 2.8 and 2.9 at week 12, and 2.7 and 2.2 at week 24. CONCLUSIONS: Lubiprostone was well tolerated in the pediatric population. The incidence of TEAEs was comparable to that observed in previous clinical trials and in adults.

Temporal, Spatial, and Epidemiologic Relationships of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Gene Cycle Thresholds: A Pragmatic Ambi-directional Observation.

Prospective serial sampling of 70 patients revealed clinically relevant cycle thresholds (Ct) occurring 9, 26, and 36 days after symptom onset. Race, gender, and corticosteroids apparently did not influence RNA positivity. In a retrospective analysis of 180 patients, initial Ct did not correlate with requirements for admission or intensive care.

Immobilized metal affinity chromatography optimization for poly-histidine tagged proteins.

Immobilized metal affinity chromatography (IMAC) is a technique primarily used in research and development laboratories to purify proteins containing engineered histidine tags. Although this type of chromatography is commonly used, it can be problematic as differing combinations of resins and metal chelators can result in highly variable chromatographic performance and product quality results. To generate a robust IMAC purification process, the binding differences of resin and metal chelator combinations were studied by generating breakthrough curves with a poly-histidine tagged bispecific protein. The optimal binding combination was statistically analyzed to determine the impact of chromatographic parameters on the operation. Additionally, equilibrium uptake isotherms were created to further elucidate the impact of chromatographic parameters on the binding of protein. It was found that for protein expressed in CHO cells, Millipore Sigma's Fractogel EMD Chelate (M) charged with Zn2+ and GE's pre-charged Ni Sepharose Excel displayed the highest binding capacities. When the protein was expressed in HEK-293, GE's IMAC Sepharose 6 Fast Flow charged with either Co2+ or Zn2+ bound the greatest amount of protein. The study further identified the metal binding capacity of the resin lot, the protein capacity to which the resin is loaded, and the ratio of poly-histidine tag residues on the protein all impacted the chromatographic performance and product quality. These findings enabled the development of a robust and scalable process. The CHO expressed cell culture product was directly loaded at a high capacity onto variable metal binding affinity Fractogel EMD Chelate (M). A 250 mM imidazole elution condition ensured the product contained monomeric 4 and 6-histidine tagged bispecific proteins. The optimized IMAC process conditions determined in this study can be applied to a wide variety of poly-histidine tagged proteins in research and development laboratories as various poly-histidine tagged proteins of differing molecular weights and formats expressed in either HEK-293 or CHO cells were successfully purified.

Sustainably reducing device utilization and device-related infections with DeCATHlons, device alternatives, and decision support.

Engagement of frontline staff, along with senior leadership, in competition-style healthcare-associated infection reduction efforts, combined with electronic clinical decision support tools, appeared to reduce antibiotic regimen initiations for urinary tract infections (P = .01). Mean monthly standardized infection and device utilization ratios also decreased (P < .003 and P < .0001, respectively).

A Phase II Trial of Safety, Tolerability and Immunogenicity of V114, a 15-Valent Pneumococcal Conjugate Vaccine, Compared With 13-Valent Pneumococcal Conjugate Vaccine in Healthy Infants.

BACKGROUND: Pneumococcal disease remains a public health priority worldwide. This phase 2 study (V114-008; NCT02987972; EudraCT 2016-001117-25) compared safety and immunogenicity of 2 clinical lots of V114 (investigational 15-valent pneumococcal vaccine: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F, 19A, 22F*, 23F, 33F*) to 13-valent pneumococcal conjugate vaccine (PCV13) in healthy infants (*serotypes unique to V114). METHODS: Healthy infants 6-12 weeks old were randomized to receive a 4-dose regimen of V114 Lot 1, V114 Lot 2 or PCV13 at 2, 4, 6 and 12-15 months old. Adverse events were evaluated after each dose. Primary immunogenicity endpoint was to demonstrate noninferiority of V114 Lot 1 and V114 Lot 2 relative to PCV13 based on proportion of infants achieving serotype-specific IgG concentration ≥0.35 µg/mL for 13 serotypes shared with PCV13 at 1 month postdose 3 (PD3). Serotype-specific IgG geometric mean concentrations (GMCs) for all 15 V114 serotypes were measured at PD3, predose 4 and 1 month postdose 4 (PD4). RESULTS: Overall, 1044 of 1051 randomized infants received ≥1 dose of vaccine (V114 Lot 1 [n = 350], V114 Lot 2 [n = 347] or PCV13 [n = 347]). Adverse events were generally comparable across groups. At PD3, both V114 lots met noninferiority criteria for all 13 serotypes shared with PCV13. IgG GMCs were comparable among V114 and PCV13 recipients at PD3 and PD4. Serotype 3 responses were higher following receipt of V114 than PCV13. Both V114 lots induced higher GMCs than PCV13 to the 2 unique V114 serotypes. CONCLUSIONS: Immunogenicity of both V114 lots was noninferior to PCV13 for all 13 shared serotypes between the 2 vaccines and displayed comparable safety and tolerability profiles to PCV13.

Simultaneous Detection of 13 Allergens in Thermally Processed Food Using Targeted LC-MS/MS Approach.

Food allergy is a major concern for public health and food industries. Because of the large numbers of food ingredients to be tested, MS is considered an alternative to existing techniques in terms of high selectivity, sensitivity, and capability to analyze multiple allergens simultaneously. In this study, we developed the method for monitoring significant peptides derived from 13 food allergens (milk, eggs, cod, shrimp, lobster, almonds, brazil nuts, cashew nuts, hazelnuts, walnuts, peanuts, wheat, and soybeans) and evaluated it in thermally processed foods (bread, cookie, fried fish, and frozen pasta). To select significant peptides to monitor, we used a bioinformatics-based approach and experimental confirmatory analysis. It was demonstrated that the developed method could detect target food ingredients from thermally processed foods successfully.

Semi-Automated Visualization and ANalysis of Trends: A "SAVANT" for Facilitating Antimicrobial Stewardship Using Antistaphylococcal Resistance and Consumption as a Prototype.

BACKGROUND: Governments and health care regulators now require hospitals and nursing homes to establish programs to monitor and report antimicrobial consumption and resistance. However, additional resources were not provided. We sought to develop an approach for monitoring antimicrobial resistance and consumption that health care systems can implement with minimal added costs or modifications to existing diagnostic and informatics infrastructure. METHODS: Using (1) the electronic laboratory information system of a nationwide managed care network, (2) the 3 most widely used commercial microbiology diagnostic platforms, and (3) Staphylococcus aureus, one of the most common causes of infections worldwide, as a prototype, we validated the approach dubbed "SAVANT" for Semi-Automated Visualization and ANalysis of Trends. SAVANT leverages 3 analytical methods (time series analysis, the autoregressive integrated moving average, and generalized linear regression) on either commercial or open source software to report trends in antistaphylococcal use and resistance. RESULTS: All laboratory results from January 2010 through December 2015 from an annual average of 9.2 million health care beneficiaries were queried. Inpatient and outpatient prescription rates were calculated for 8 key antistaphylococcal compounds. Trends and relationships of antistaphylococcal consumption and resistance among 81 840 unique S. aureus isolates from >6.5 million cultures were revealed. CONCLUSIONS: Using existing or freely available resources, SAVANT was successfully implemented across a complex and geographically dispersed 280-hospital network, bridging a critical gap between medical informatics, large-scale data analytics, and mandatory reporting of health care quality metrics.

Trends in Utilization of Outpatient Respiratory Syncytial Virus Prophylaxis with Palivizumab among Medicaid- and Commercially Insured Infants.

INTRODUCTION: Palivizumab is indicated for the prevention of respiratory syncytial virus (RSV) infection in high-risk children. Previous palivizumab utilization studies examined prior authorization claims but did not examine utilization within insured populations as a whole. This study describes outpatient palivizumab utilization trends and characterizes high-risk infants receiving palivizumab within Medicaid- and commercially insured populations. METHODS: Infants born July 1, 2003 through June 30, 2013 were identified in the MarketScan® Multistate Medicaid and Commercial claims databases. Infants with ≥ 18 months of continuous medical insurance enrollment with pharmacy benefits after birth and evidence of chronic lung disease of prematurity (CLDP), congenital heart disease (CHD), or preterm birth without CLDP or CHD were studied. Palivizumab use and demographic and clinical characteristics were measured in infant subgroups. Outpatient palivizumab utilization rates were calculated for each seasonal year (July-June) and for each infant subgroup. RESULTS: In total, 29,350 (2.1%) Medicaid-insured and 9589 (2.5%) commercially insured infants received palivizumab and had CLDP, CHD, or were born at < 37 weeks gestational age (wGA). Infants with CLDP (82%) and those < 29 wGA (78%) had the highest utilization. Decreases in utilization rates between the 2003-2004 and 2012-2013 seasons were seen among Medicaid-insured infants born at 29-36 wGA (all P < .0001), and commercially insured infants born at 31-32 wGA (P < .0001), 33-34 wGA (P = .055), 35-36 wGA (P < .0001), and with CHD (P = .003). Utilization by month was consistent across subgroups among Medicaid- and commercially insured infants, with most doses administered from November to March. CONCLUSION: Palivizumab use is targeted to a small percentage of infants who are at highest risk of hospitalization for RSV disease. Utilization declined in recent years in both Medicaid- and commercially insured infant groups. Most palivizumab doses were administered from November to March, with most infants receiving ≤ 5 doses. FUNDING: AstraZeneca.

Analysis of Serial Isolates of mcr-1-Positive Escherichia coli Reveals a Highly Active ISApl1 Transposon.

The emergence of a transferable colistin resistance gene (mcr-1) is of global concern. The insertion sequence ISApl1 is a key component in the mobilization of this gene, but its role remains poorly understood. Six Escherichia coli isolates were cultured from the same patient over the course of 1 month in Germany and the United States after a brief hospitalization in Bahrain for an unconnected illness. Four carried mcr-1 as determined by real-time PCR, but two were negative. Two additional mcr-1-negative E. coli isolates were collected during follow-up surveillance 9 months later. All isolates were analyzed by whole-genome sequencing (WGS). WGS revealed that the six initial isolates were composed of two distinct strains: an initial ST-617 E. coli strain harboring mcr-1 and a second, unrelated, mcr-1-negative ST-32 E. coli strain that emerged 2 weeks after hospitalization. Follow-up swabs taken 9 months later were negative for the ST-617 strain, but the mcr-1-negative ST-32 strain was still present. mcr-1 was associated with a single copy of ISApl1, located on a 64.5-kb IncI2 plasmid that shared >95% homology with other mcr-1 IncI2 plasmids. ISApl1 copy numbers ranged from 2 for the first isolate to 6 for the final isolate, but ISApl1 movement was independent of mcr-1 Some movement was accompanied by gene disruption, including the loss of genes encoding proteins involved in stress responses, arginine catabolism, and l-arabinose utilization. These data represent the first comprehensive analysis of ISApl1 movement in serial clinical isolates and reveal that, under certain conditions, ISApl1 is a highly active IS element whose movement may be detrimental to the host cell.