High risk of necrotising enterocolitis in term-born neonates with CHD delivered by caesarean section: a case-control study.

OBJECTIVE: Necrotising enterocolitis is linked with altered intestinal microbiota, and caesarean birth is associated with imbalance of newborn intestinal microbiome. We aimed to investigate the role of delivery mode (vaginal or caesarean) and gestational age in the development of necrotising enterocolitis among term-born neonates (≥ 37 weeks) with CHD. METHODS: Case-control study. We studied all newborns with CHD who underwent cardiac surgery during the neonatal (≤ 28 days of age) period, between 2007 and 2017. Totally, 60 cases of necrotising enterocolitis were matched (by year of birth and type of congenital heart lesion) with 180 controls (1:3 ratio). Multivariable conditional logistic regression was used to assess the study question. RESULTS: The overall prevalence of necrotising enterocolitis was 6.3% in term-born newborns with CHD. Neonates with a left-ventricular outflow tract lesion or single ventricle lesion accounted for 55% (n = 33) of cases. 62% (n = 37) cases were in the modified Bell's stage 2 or more for necrotising enterocolitis classification. In multivariable modelling, gestational age at birth was not associated with the development of necrotising enterocolitis [adjusted odds ratio per week increase, 95% confidence interval: 1.20 (0.90-1.60)]. Birth by caesarean delivery (compared to vaginal) was strongly associated with development of necrotising enterocolitis [adjusted odds ratio (95% confidence interval): 2.64 (1.31-5.29)]. We failed to identify an association between preoperative enteral nutrition and necrotising enterocolitis. CONCLUSION: This study showed a high risk of necrotising enterocolitis in newborns with critical CHD born via caesarean. This information is important given the high prevalence of planned birth by caesarean in newborns with CHD.

Potential benefits of prenatal diagnosis of TGA in Australia may be outweighed by the adverse effects of earlier delivery: likely causation and potential solutions.

OBJECTIVE: Prenatal diagnosis of transposition of great arteries (TGA) is expected to improve postoperative outcomes after neonatal arterial switch operation (ASO); however, published reports give conflicting results. We aimed to determine the association between prenatal diagnosis and early postoperative outcomes after neonatal ASO. METHODS: Cohort study involving 243 newborns who underwent ASO (70% prenatally diagnosed) between 2010 and 2019. Multivariable regression was used to determine the association between prenatal diagnosis and (a) birth characteristics and (b) postoperative outcomes. RESULTS: Gestational age and birthweight centile were lower and small-for-gestational-age more common (11.8% vs 1.4%) in those diagnosed prenatally. Among births which followed labour induction or prelabour caesarean, prenatal diagnosis was associated with earlier gestation at birth (mean (SD), 38.5 (1.6) vs 39.2 (1.4), p=0.01). Among births which followed spontaneous labour, prenatal diagnosis was associated with earlier gestation at labour onset (38.2 (1.8) vs 39.2 (1.4), p=0.01). Prenatal diagnosis was associated with longer postoperative mechanical ventilation (incidence rate ratio 1.74, 95% CI 1.37 to 2.21), intensive care (1.70, 1.31 to 2.21) and hospital length of stay (1.37, 1.14 to 1.66) after ASO. Gestational age mediated up to 60% of the effect of prenatal diagnosis on postoperative outcomes. CONCLUSION: Among newborns undergoing ASO for TGA, prenatal diagnosis is associated with poorer early postoperative outcomes. In addition to minimising iatrogenic factors (such as planned births) resulting in earlier births, evaluation of other dynamics following a prenatal diagnosis which may result in poor fetal growth and earlier onset of spontaneous labour is important.

Experiences in aligning WHO SMART guidelines to classification and terminology standards.

OBJECTIVES: Digital adaptation kits (DAKs) distill WHO guidelines for digital use by representing them as workflows, data dictionaries and decision support tables. This paper aims to highlight key lessons learnt in coding data elements of the antenatal care (ANC) and family planning DAKs to standardised classifications and terminologies (CATs). METHODS: We encoded data elements within the ANC and family planning DAKs to standardised CATs from the WHO CATs and other freely available CATs. RESULTS: The coding process demonstrated approaches to refine the data dictionaries and enhance alignment between data elements and CATs. DISCUSSION: Applying CATs to WHO clinical and public health guidelines can ensure that recommendations are operationalised in a digital system with appropriate consistency and clarity. This requires a multidisciplinary team and careful review to achieve conceptual equivalence between data elements and standardised terminologies. CONCLUSION: The systematic translation of guidelines into digital systems provides an opportunity for leveraging CATs; however, this approach needs further exploration into its implementation in country contexts and transition into machine-readable components.

Fluid Accumulation in Mechanically Ventilated, Critically Ill Children: Retrospective Cohort Study of Prevalence and Outcome.

OBJECTIVES: To describe the prevalence, patterns, explanatory variables, and outcomes associated with fluid accumulation (FA) in mechanically ventilated children. DESIGN: Retrospective cohort study. SETTING: Tertiary PICU. PATIENTS: Children mechanically ventilated for greater than or equal to 24 hours. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Between July 2016 and July 2021, 1,636 children met eligibility criteria. Median age was 5.5 months (interquartile range [IQR], 0.7-46.5 mo), and congenital heart disease was the most common diagnosis. Overall, by day 7 of admission, the median maximum cumulative FA, as a percentage of estimated admission weight, was 7.5% (IQR, 3.3-15.1) occurring at a median of 4 days after admission. Overall, higher FA was associated with greater duration of mechanical ventilation (MV) (mean difference, 1.17 [95% CI, 1.13-1.22]; p < 0.001]), longer intensive care length of stay (LOS) (mean difference, 1.16 [95% CI, 1.12-1.21]; p < 0.001]), longer hospital LOS (mean difference, 1.19 [95% CI, 1.13-1.26]; p < 0.001]), and increased mortality (odds ratio, 1.31 [95% CI, 1.08-1.59]; p = 0.005). However, these associations depended on the effects of children with extreme values, and there was no increase in risk up to 20% FA, overall, in children following cardiopulmonary bypass and in children in the general ICU. When excluding children with maximum FA of >10%, there was no association with duration of MV (mean difference, 0.99 [95% CI, 0.94-1.04]; p = 0.64) and intensive care or hospital LOS (mean difference, 1.01 [95% CI, 0.96-1.06]; p = 0.70 and 1.01 [95% CI, 0.95-1.08]; 0.79, respectively) but an association with reduced mortality 0.71 (95% CI, 0.53-0.97; p = 0.03). CONCLUSIONS: In mechanically ventilated critically ill children, greater maximum FA was associated with longer duration of MV, intensive care LOS, hospital LOS, and mortality. However, these findings were driven by extreme values of FA of greater than 20%, and up to 10%, there was reduced mortality and no signal of harm.

Changes in on-time vaccination following the introduction of an electronic immunization registry, Tanzania 2016-2018: interrupted time-series analysis.

BACKGROUND: Digital health interventions (DHI) have the potential to improve the management and utilization of health information to optimize health care worker performance and provision of care. Despite the proliferation of DHI projects in low-and middle-income countries, few have been evaluated in an effort to understand their impact on health systems and health-related outcomes. Although more evidence is needed on their impact and effectiveness, the use of DHIs among immunization programs has become more widespread and shows promise for improving vaccination uptake and adherence to immunization schedules. METHODS: Our aim was to assess the impact of an electronic immunization registry (EIR) using an interrupted time-series analysis to analyze the effect on proportion of on-time vaccinations following introduction of an EIR in Tanzania. We hypothesized that the introduction of the EIR would lead to statistically significant changes in vaccination timeliness at 3, 6, and > 6 months post-introduction. RESULTS: For our primary analysis, we observed a decrease in the proportion of on-time vaccinations following EIR introduction. In contrast, our sensitivity analysis estimated improvements in timeliness among those children with complete vaccination records. However, we must emphasize caution interpreting these findings as they are likely affected by implementation challenges. CONCLUSIONS: This study highlights the complexities of using digitized individual-level routine health information system data for evaluation and research purposes. EIRs have the potential to improve vaccination timeliness, but analyses using EIR data can be complicated by data quality issues and inconsistent data entry leading to difficulties interpreting findings.

Transitioning to Digital Systems: The Role of World Health Organization's Digital Adaptation Kits in Operationalizing Recommendations and Interoperability Standards.

INTRODUCTION: The transition from paper to digital systems requires quality assurance of the underlying content and application of data standards for interoperability. The World Health Organization (WHO) developed digital adaptation kits (DAKs) as an operational and software-neutral mechanism to translate WHO guidelines into a standardized format that can be more easily incorporated into digital systems. METHODS: WHO convened health program area and digital leads, reviewed existing approaches for requirements gathering, mapped to established standards, and incorporated research findings to define DAK components. RESULTS: For each health domain area, the DAKs distill WHO guidelines to specify the health interventions, personas, user scenarios, business process workflows, core data elements mapped to terminology codes, decision-support logic, program indicators, and functional and nonfunctional requirements. DISCUSSION: DAKs aim to catalyze quality of care and facilitate data use and interoperability as part of WHO's vision of SMART (Standards-based, Machine-readable, Adaptive, Requirements-based, and Testable) guidelines. Efforts will be needed to strengthen a collaborative approach for the uptake of DAKs within the local digital ecosystem and national health policies.

Characteristics, management and changing incidence of children with empyema in a paediatric intensive care unit.

AIM: Paediatric intensive care unit (PICU) admissions for empyema increased following the 13-valent pneumococcal conjugate vaccine (PCV13). We describe the clinical characteristics, management and outcomes for children with empyema and compare incidence before and after PCV13. METHODS: Retrospective study of patients <18 years admitted to The Royal Children's Hospital Melbourne PICU with empyema between January 2016 and July 2019. We investigated the incidence of empyema during two time periods: 2007-2010 (pre-PCV13) and 2016-2019 (post-PCV13). RESULTS: Seventy-one children (1.9% of all PICU admissions) were admitted to PICU with empyema between 2016 and 2019. Sixty-one (86%) had unilateral disease, 11 (16%) presented with shock and 44 (62%) were ventilated. Streptococcus pneumoniae and group A Streptococcus were the most commonly identified pathogens. Forty-five (63%) were managed with video-assisted thoracoscopic surgery (VATS). There was a 31% reduction in empyema hospitalisations as a proportion of all hospitalisations (IRR 0.69, 95% CI 0.59-0.8), but a 2.8-fold increase in empyema PICU admissions as a proportion of all PICU admissions (95% CI 2.2-3.5, P < 0.001). For the PICU cohort, this was accompanied by reduction in PIM2 probability of death (median 1% vs. 1.9%, P = 0.02) and duration of intubation (median 69 h vs. 126.5 h, P = 0.045). CONCLUSIONS: In children with empyema in PICU 62% required ventilation, 16% had features of shock and 63% received VATS. Empyema admissions, as a proportion of all PICU admissions, increased in the era post-PCV13 compared to pre-PCV13 despite no increase in illness severity at admission.

Recurrent Extubation Failure Following Neonatal Cardiac Surgery Is Associated with Increased Mortality.

Extubation failure (EF) following neonatal cardiac surgery is associated with increased mortality. Neonates who experienced EF twice or more (recurrent EF) may have worse outcomes than those who have a single EF or no-EF. The aims of this study are to investigate the in hospital mortality for neonates with recurrent EF compared to those with single or no-EF, and determine factors associated with recurrent EF. Neonates' ≤ 28 days who underwent cardiac surgery from January 2008 to December 2019 were included. EF was defined as unplanned reintubation within 72 h after a planned extubation. 1187 (18 recurrent EF, 84 single EF and 1085 no-EF) neonates were included. Recurrent EF occurred in 18 (17.6%) of 102 neonates undergoing a second extubation. The median time (IQR) to reintubation after the first and second extubations were similar, being 20.9 (3.3-45.2) versus 19.4 (5.5-47) h. The reason for a second-time EF was respiratory in 39% and cardiovascular in 33%. Recurrent EF and single EF was associated with increased mortality (odds ratio, 95% confidence interval (CI) 23.5, 6.9-79.9) and (odds ratio, 95% CI 5.2, 2.3-12.0) compared to no-EF. Based on the final model with risk adjustment, predicted mortality was 29.0% in recurrent EF, 6.5% in single EF, and 1.2% in no-EF. First-time EF due to cardiovascular compromise was associated with recurrent EF (odds ratio, 95% CI 3.1, 1.0-9.7). This study confirmed that patients with recurrent EF have a high morality. Neonates with a cardiovascular reason for first-time EF are more likely to have a recurrent EF than those with other causes.

Characteristics and outcomes of children receiving intensive care therapy within 12 hours following a medical emergency team event.

Objectives: To describe characteristics and outcomes of children requiring intensive care therapy (ICT) within 12 hours following a medical emergency team (MET) event. Design: Retrospective cohort study. Setting: Quaternary paediatric hospital. Patients: Children experiencing a MET event. Measurements and main results: Between July 2017 and March 2019, 890 MET events occurred in 566 patients over 631 admissions. Admission to intensive care followed 183/890 (21%) MET events. 76/183 (42%) patients required ICT, defined as positive pressure ventilation or vasoactive support in intensive care, within 12 hours. Older children had a lower risk of requiring ICT than infants aged < 1 year (age 1-5 years [risk difference, -6.4%; 95% CI, -11% to -1.6%; P = 0.01] v age > 5 years [risk difference, -8.0%; 95% CI, -12% to -3.8%; P < 0.001]), while experiencing a critical event increased this risk (risk difference, 16%; 95% CI, 3.3-29%; P = 0.01). The duration of respiratory support and intensive care length of stay was approximately double in patients requiring ICT (ratio of geometric means, 2.0 [95% CI, 1.4-3.0] v 2.1 [95% CI, 1.5-2.8]; P < 0.001) and the intensive care mortality increased (risk difference, 9.6%; 95% CI, 2.4-17%; P = 0.01). Heart rate, oxygen saturation and respiratory rate were the most commonly measured vital signs in the 6 hours before the MET event. Conclusions: Approximately one-fifth of MET events resulted in intensive care admission and nearly half of these required ICT within 12 hours. This group had greater duration of respiratory support, intensive care and hospital length of stay, and higher mortality. Age < 1 year and a critical event increased the risk of ICT.

Interhospital transport of children with bronchiolitis by a statewide emergency transport service.

Objective: To investigate the rate of interhospital emergency transport for bronchiolitis and intensive care admission following the introduction of high flow nasal cannula and standardised paediatric observation and response charts. Design: Retrospective cohort study. Setting: A statewide paediatric intensive care transport service and its two referral paediatric intensive care units (PICUs) in Victoria, Australia. Participants: Children less than 2 years old emergently transported with bronchiolitis during two time periods: 2008-2012 and 2015-2019. Main outcome measures: Incidence rates of bronchiolitis transport episodes, PICU admissions and respiratory support. Results: 802 children with bronchiolitis were transported during the study period, 233 in the first period (2008-2012) and 569 in the second period (2015-2019). The rate of interhospital transport for bronchiolitis increased from 32.9 to 71.8 per 100 000 children aged 0-2 years. The population-adjusted rate of PICU admission increased from 16.2 to 36.6 per 100 000 children aged 0-2 years. Metropolitan hospitals were the predominant referral source and this increased from 60.1% of transports to 78.6% (P < 0.001). In children admitted to a PICU, the administration of high flow nasal cannula during transport increased significantly from 1.7% to 75.9% (P < 0.001) and a concomitant reduction in continuous positive airway pressure and mechanical ventilation occurred (40-12.4% and 27-6.9% respectively; P < 0.001). The proportion of mechanical ventilation as well as PICU and hospital length of stay decreased over time. Conclusions: The population-adjusted rate of interhospital transport and admission to the PICU for bronchiolitis increased over time. This occurred despite a lower rate of non-invasive and invasive mechanical ventilation during transport and in the PICU.

Ten years of severe respiratory syncytial virus infections in a tertiary paediatric intensive care unit.

AIM: To describe the epidemiology and treatment of respiratory syncytial virus (RSV) infection in a tertiary paediatric intensive care unit (PICU), including the clinical presentations, comorbidities, respiratory support required, costs and outcomes. METHODS: This study was an analysis of a database for all children with RSV infections admitted to the PICU in Melbourne between 2005 and 2015. RESULTS: A total of 604 episodes of community-acquired RSV infections were analysed, and the median age of children was 4 months (interquartile range 2-14 months); 94% of cases had lower respiratory tract infection, principally bronchiolitis, and 8.9% presented with extrapulmonary features. Respiratory support included humidified high-flow nasal cannula oxygen therapy (76% of patients since its introduction in 2011), non-invasive ventilation (41%) and intubation and mechanical ventilation (32%). Almost half (n = 270; 45%) had one or more pre-existing comorbid condition. Risk factors for intubation and mechanical ventilation were presence of comorbidities (odds ratio 1.97; confidence interval 1.39-2.79, P < 0.001) and transfer from an external hospital (odds ratio 1.82; confidence interval 1.58-2.57, P < 0.001). Of the children without pre-existing comorbidities, 25% required intubation and mechanical ventilation. Following the introduction of humidified high-flow nasal cannula oxygen therapy, the number of annual PICU admissions for RSV infection doubled; however, the number of children requiring intubation remained unchanged. The median length of intensive care unit stay was 3.7 days and further hospital stay was 3.6 days, and the average cost per case was approximately AU$20000. CONCLUSIONS: RSV infection carries a high burden in PICU, in bed-days and cost. Chronic comorbidities and transfer from a peripheral hospital were associated with a higher rate of need for mechanical ventilation.

The impact of an integrated electronic immunization registry and logistics management information system (EIR-eLMIS) on vaccine availability in three regions in Tanzania: A pre-post and time-series analysis.

BACKGROUND: Since 2016, the Government of Tanzania has been implementing TImR, an integrated Electronic Immunization registry-logistics management information system (EIR-LMIS) that includes stock notifications. The objective of this study is to estimate the impact of this intervention on vaccine availability. METHODS: Monthly stock-out data were collected from paper registers at facilities, an Excel-based system at districts, and the new system (TImR) across all 924 health facilities in Arusha, Tanga and Kilimanjaro Regions. Six months of stockout rates pre- and post-introduction, by antigen, were compared via a two-way analysis of variance (ANOVA). A mixed-effects logistic regression model with the TImR data identified predictors of vaccine availability across antigens. FINDINGS: Post-introduction, ANOVA models estimated that overall stock-out rates declined from a monthly average of 7.1% to 2.1% (p < 0.01). Three specific vaccines had fewer stock-outs; OPV's monthly average dropped from 12.5% to 2.1% (p < 0.01), MR from 9.4% to 1.0% (p < 0.01) and DTP-HepB-HiB from 8.1% to 1.7% (p < 0.01). In the mixed-effects logistic regression model, controlling for antigen, odds of stock-out were 4.1% (95% CI: 3.3 - 4.9) lower for each week of tenure. Compared to DTP-HepB-HiB vaccine, odds of BCG vaccine being stocked out were 4.31 as high (95% CI: 3.1 - 5.0). The odds of being stocked-out were 29.7% lower for PCV (95% CI: 8.8 - 45.8) and 26.6% (95% CI: 3.4 - 44.1) lower for rotavirus vaccines compared to DTP-HepB-HiB. The odds of stock out were 37.7% lower for MR vaccine than DTP-HepB-HiB (95% CI: 18.1 - 52.6). CONCLUSIONS: Tanzania's integrated EIR-eLMIS may increase vaccine availability compared to its paper and Excel based system. Post-introduction of an eLMIS, the odds of a vaccine stock-out reduced over time. Further research could determine the impact of this intervention on vaccine wastage and replenishment response times.

Cathepsin D: Removal strategy on protein A chromatography, near real time monitoring and characterisation during monoclonal antibody production.

Monoclonal antibody (mAb) fragmentation is a well-known degradation pathway that results in product loss and can significantly impact product quality, efficacy, or even cause immunogenic reactions, thus potentially endangering patients' health. It is recognised that residual proteases present among host cell proteins (HCPs) such as those expressed by Chinese Hamster Ovary (CHO) can induce fragmentation, and failure of their complete removal during downstream processing could cause fragmentation during mAb production and in the final drug product. We identified, using a protease inhibitor screen, an aspartic protease that contributes to proteolytic fragmentation of partially purified mAbs in multiple projects. Subsequent LC-MS analysis indicated that cathepsin D, a typical aspartic protease, was responsible for the observed fragmentation of in-process samples. To address the issue, an alternative chromatography wash was implemented at the capture step and has been demonstrated to be an effective and scalable solution to mitigate the residual cathepsin D associated fragmentation risk. Furthermore, a near real time targeted mass spectrometry method has been developed to proactively monitor the presence of cathepsin D during upstream and downstream process. Our approach demonstrated an emerging HCP mitigation strategy through integrated upstream and downstream involvement and holds great promise for a range of future applications.

Investigation of cathepsin D-mAb interactions using a combined experimental and computational tool set.

Cathepsin D has been identified as a challenge to remove in downstream bioprocessing of monoclonal antibodies (mAbs) due to interactions with some mAbs. This study focused on investigating the mechanisms of interaction between cathepsin D and two industrial mAbs using a combined experimental and computational approach. Surface plasmon resonance was used to study the impact of pH and salt concentration on these protein-protein interactions. While salt had a moderate effect on the interactions with one of the mAbs, the other mAb demonstrated highly salt-dependent association behavior. Cathepsin D binding to the mAbs was also seen to be highly pH dependent, with operation at pH 9 resulting in a significant decrease in the binding affinity. Protein-protein docking simulations identified three interaction sites on both mAbs; near the complementarity determining region (CDR), in the hinge, and in the CH 3 domain. In contrast, only one face of cathepsin D was identified to interact with all the three sites on the mAbs. Surface property analysis revealed that the binding regions on the mAbs contained strong hydrophobic clusters and were predominantly negatively charged. In contrast, the binding site on cathepsin D was determined to be highly positively charged and hydrophobic, indicating that these protein-protein interactions were likely due to a combination of hydrophobic and electrostatic interactions. Finally, covalent crosslinking coupled with mass spectrometry was used to validate the docking predictions and to further investigate the regions of interaction involved in mAb-cathepsin D binding. A strong agreement was observed between the two approaches, and the CDR loops were identified to be important for cathepsin D interactions. This study establishes a combined experimental and computational platform that can be used to probe mAb-host cell protein (HCP) interactions of importance in biomanufacturing.

Redefining vaccination coverage and timeliness measures using electronic immunization registry data in low- and middle-income countries.

Vaccine coverage is routinely used as a performance indicator for immunization programs both at local and global levels. For many national immunization programs, there are challenges with accurately estimating vaccination coverage based on available data sources, however an increasing number of low- and middle-income countries (LMICs) have begun implementing electronic immunization registries to replace health facilities' paper-based tools and aggregate reporting systems. These systems allow for more efficient capture and use of routinely reported individual-level data that can be used to calculate dose-specific and cohort vaccination coverage, replacing the commonly used aggregate routine health information system data. With these individual-level data immunization programs have the opportunity to redefine performance measures to enhance programmatic decision-making at all levels of the health system. In this commentary, we discuss how measures for assessing vaccination status and program performance can be redefined and recalculated using these data when generated at the health facility level and the implications of the use and availability of electronic individual-level data.

Changing Risk of In-Hospital Cardiac Arrest in Children Following Cardiac Surgery in Victoria, Australia, 2007-2016.

BACKGROUND: Reported incidence of in hospital cardiac arrest (IHCA) after paediatric cardiac surgery varies between 3-4% in high income countries and this risk may have changed over time. We sought to examine this trend in detail. METHODS: A retrospective observational study of 3,781 children who underwent 4,938 cardiac surgeries between 1 January 2007 and 31 December 2016 in a tertiary children's hospital. IHCA was defined as cessation of cardiac mechanical activity requiring cardiac massage for ≥1minute. Surgical complexity was categorised using risk adjusted congenital heart surgery (RACHS-1) category. Poisson regression was used to analyse trends for every two-year period. RESULTS: There were a total of 211 (4.3%) IHCA events after surgery. These patients were younger, more likely to have had a premature birth, have a chromosomal or genetic syndrome association and have a high surgical complexity. Overall, there was a 52% reduction in IHCA rate over 10 years: reducing from 5.4 /100 surgeries in 2007-08 to 2.6/100 surgeries in 2015-16 (p-trend=<0.001). The reduction was mainly seen in low-to-moderate risk categories (RACHS-1 categories 1-4) and not in high risk categories (RACHS-1 category 5-6). Children in high risk categories were 13.6 times more likely to experience an IHCA (compared to low risk categories). Overall hospital mortality for children suffering IHCA decreased from 42.5/100 patients in 2007-08 to 11.1/100 patients in 2015-16 (p-trend=0.037). CONCLUSIONS: The IHCA rate following cardiac surgery has more than halved over the last decade; children who experience IHCA also have lower mortality than in previous years. High risk procedures still have a substantial rate of IHCA and efforts are needed to minimise the burden further in this population.

The Hybrid Search: A Mass Spectral Library Search Method for Discovery of Modifications in Proteomics.

We present a mass spectral library-based method to identify tandem mass spectra of peptides that contain unanticipated modifications and amino acid variants. We describe this as a "hybrid" method because it combines matching both ion m/z and mass losses. The mass loss is the difference between the mass of an ion peak and the mass of its precursor. This difference, termed DeltaMass, is used to shift the product ions in the library spectrum that contain the modification, thereby allowing library product ions that contain the unexpected modification to match the query spectrum. Clustered unidentified spectra from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and Chinese hamster ovary cells were used to evaluate this method. The results demonstrate the ability of the hybrid method to identify unanticipated modifications, insertions, and deletions, which may include those due to an incomplete protein sequence database or to search settings that exclude the correct identification, in high-resolution tandem mass spectra without regard to their precursor mass. This has been made possible by indexing of the m/z value of each fragment ion and its difference in mass from its precursor ion.

Identification of an IgG CDR sequence contributing to co-purification of the host cell protease cathepsin D.

Recombinant therapeutic monoclonal antibodies (mAbs) must be purified from host cell proteins (HCPs), DNA, and other impurities present in Chinese hamster ovary (CHO) cell culture media. HCPs can potentially result in adverse clinical responses in patients and, in specific cases, have caused degradation of the final mAb product. As reported previously, residual traces of cathepsin D caused particle formation in the final product of mAb-1. The current work was focused on identification of a primary sequence in mAb-1 responsible for the binding and consequent co-purification of trace levels of CHO cathepsin D. Surface plasmon resonance (SPR) was used to detect binding between immobilized CHO cathepsin D and a panel of mAbs. Out of 13 mAbs tested, only mAb-1 and mAb-6 bound to cathepsin D. An LYY motif in the HC CDR2 was common, yet unique, to only these two mAbs. Mutation of LYY to AAA eliminated binding of mAb-1 to cathepsin D providing confirmation that this sequence motif was involved in the binding to CHO cathepsin D. Interestingly, the binding between mAb-1 and cathepsin D was weaker than that of mAb-6, which may be related to the fact that two aspartic acid residues near the LYY motif in mAb-1 are replaced with neutral serine residues in mAb-6. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:140-145, 2017.

Different Regions of Plasmodium falciparum Erythrocyte-Binding Antigen 175 Induce Antibody Responses to Infection of Varied Efficacy.

BACKGROUND: Increasing evidence suggests that antibodies against merozoite proteins involved in Plasmodium falciparum invasion into the red blood cell play an important role in clinical immunity to malaria. Erythrocyte-binding antigen 175 (EBA-175) is the best-characterized P. falciparum invasion ligand, reported to recognize glycophorin A on the surface of erythrocytes. Its protein structure comprises 6 extracellular regions. Whereas region II contains Duffy binding-like domains involved in the binding to glycophorin A, the functional role of regions III-V is less clear. METHODS: We developed a novel cytometric bead array for assessment of antigen-specific antibody concentration in plasma to evaluate the efficacy of immune responses to different regions of EBA-175 and associations between antibody levels with protection from symptomatic malaria in a treatment-reinfection cohort study. RESULTS: We found that while antibodies to region II are highly abundant, circulating levels as low as 5-10 µg/mL of antibodies specific for region III or the highly conserved regions IV-V predict strong protection from clinical malaria. CONCLUSIONS: These results lend support for the development of conserved regions of EBA-175 as components in a combination of a malaria vaccine.