Antiviral susceptibility of influenza viruses is monitored by the World Health Organization Global Influenza Surveillance and Response System. This study describes a global analysis of the susceptibility of influenza viruses to neuraminidase (NA) inhibitors (NAIs, oseltamivir, zanamivir, peramivir, laninamivir) and the cap-dependent endonuclease inhibitor (CENI, baloxavir) for three periods (May to May for 2020-2021, 2021-2022 and 2022-2023). In particular, global influenza activity declined significantly in 2020-2021 and 2021-2022 when compared to the pre-pandemic period of COVID-19. Combined phenotypic and NA sequence-based analysis revealed that the global frequency of seasonal influenza viruses with reduced or highly reduced inhibition (RI/HRI) by NAIs remained low, 0.09% (2/2224), 0.12% (27/23465) and 0.23% (124/53917) for 2020-2021, 2021-2022 and 2022-2023, respectively. As in previous years, NA-H275Y in A(H1N1)pdm09 viruses was the most frequent substitution causing HRI by oseltamivir and peramivir. Sequence-based analysis of polymerase acidic (PA) protein supplemented with phenotypic testing revealed low global frequencies of seasonal influenza viruses with reduced susceptibility (RS) to baloxavir, 0.07% (1/1376), 0.05% (9/18380) and 0.12% (48/39945) for 2020-2021, 2021-2022 and 2022-2023, respectively; commonly associated substitutions were PA-I38T/M/L. In Japan, the rate was 3.3% (16/488) during 2022-2023, with 11 A(H3N2) viruses having PA-I38T/M substitutions. For zoonotic viruses, 2.7% (3/111) contained substitutions, one each NA-H275Y, NA-S247N and NA-N295S, associated with RI/HRI NAI phenotypes, and none contained PA substitutions associated with RS to baloxavir. In conclusion, the great majority of seasonal and zoonotic influenza viruses remained susceptible to NAIs and CENI baloxavir.

Achieving the Sustainable Development Goal of reducing child mortality to <25 deaths per 1000 live births by 2030 requires strategies to prevent diarrheal disease-related morbidity and mortality. Accurate etiological diagnosis is essential. This study used postmortem diagnostics to investigate the contribution of diarrhea to under-5 mortality and examine co-morbidities and co-infections in Africa and South Asia. Child Health and Mortality Prevention Surveillance (CHAMPS) generates data on child deaths through minimally invasive tissue sampling, clinical record review, and verbal autopsies. Multidisciplinary panels assign cause(s) of death using WHO International Classification of Diseases. This analysis included deaths among children aged 1-59 months enrolled from 18 December 2016-31 December 2023 across six African sites (Ethiopia, Mali, Kenya, Sierra Leone, Mozambique, South Africa) and Bangladesh. Of 1517 deaths assessed, diarrhea was in the causal pathway in 240 (15.8%). The proportion of diarrhea-related deaths was highest in Ethiopia (41.0%, 34/83), followed by Bangladesh, (30.0%, 3/10), Mozambique (21.7%, 56/258), Mali (17.5%, 18/103), Kenya (13.9%, 51/366), Sierra Leone (12.8%, 46/358), and South Africa (9.4%, 32/339). Diarrhea was underlying cause in 44.2% (106/240) of cases and immediate/antecedent cause in 58.3% (140/240), with some deaths involving multiple roles in the causal chain. When diarrhea was underlying cause, sepsis (33.0%) and lower respiratory infections (25.5%) were common downstream conditions; when an antecedent/immediate cause, leading underlying causes were malnutrition (64.3%) and HIV (13.6%). No pathogen was identified in 49.6% (119/240) of diarrhea-related deaths; among these, diarrhea was underlying cause in 42.9%. Among the 121 pathogen-attributed deaths, the most frequent were EAEC (34.7%), typical EPEC (15.7%), Shigella/EIEC (14.0%), ST-ETEC (12.4%), rotavirus (26.4%), and adenovirus (non-40/41: 19.0%; 40/41: 5.0%). These pathogens were frequently identified as co-infections. Diarrheal disease accounted for a substantial share of child deaths across CHAMPS sites. Reducing mortality will require preventing diarrhea and addressing key contributors such as malnutrition and HIV.

BACKGROUND: While electronic cigarettes (ECIG) may have lower toxicant delivery than cigarettes, ECIG-liquids and aerosols still contain toxicants that can potentially disrupt lung lipid homeostasis. METHODS: Participants from two studies underwent bronchoscopy and bronchoalveolar lavage (BAL). Ninety-eight participants (21-44 years old) were included in a cross-sectional study, with 17 ECIG users, 52 non-smokers, and 29 smokers. In the four-week clinical trial, 30 non-smokers were randomly assigned to use nicotine-free, flavorless ECIG or no use. A panel of 75 quantifiable lipid species and 7 lipid classes were assessed in the BAL using two tandem mass spectrometry (MS/MS) platforms. Ten cytokines and lipid-laden macrophages (LLM) were analyzed using the V-PLEX Plus Proinflam Combo 10 panel and Oil Red O staining, respectively. RESULTS: In the cross-sectional study, 43 lipids were associated with smoking status at FDR<0.1, including two between ECIG and non-smokers (PC(14:0/18:1) and PC(18:0/14:0)) in pairwise follow-up analyses (Bonferroni-adjusted p<0.017). Associations between lipid species and cotinine, inflammatory markers, including IL-1β and IL-8, and LLM were also identified, as well as differences in lipid classes between smokers and the other groups. Smokers had higher saturated lipids, including ceramide (CER), sphingomyelin (SM), and diacylglycerol (DAG) than that of non-smokers and ECIG users. No significant associations were identified in the 4-week clinical trial. CONCLUSIONS: Smoking was associated with altered lipid levels, as compared to both non-smokers and ECIG users; the majority were downregulated and ECIG effects tend to be smaller in magnitude than smoking effects, although some were different than those in the smokers group. This is a novel study of healthy individuals examining lipidomic differences between smokers, ECIG users, and non-smokers, indicating potential roles of smoking and ECIG-related lipid alterations in pulmonary disease. TRIAL REGISTRATION: The study was approved by The OSU Institutional Review Board (OSU-2015C0088) in accordance with its ethical standards, the Helsinki declaration, and the Belmont Report, and is registered on Clinicaltrials.gov (NCT02596685; 2015-11-04).

With the development of new vaccine technologies, such as mRNA vaccines, tissue studies are becoming increasingly important. Knowledge of the antigen expression amounts and where the antigen accumulates in the body is essential for designing safe and effective vaccines. Mammalian tissues present challenges in the detection and accurate quantification of target proteins because of their complexity and the lack of protocols that efficiently extract proteins with minimal sample loss. Here, we describe a protocol for the detection and accurate quantification of protein targets in commercially available snap-frozen lung, liver, kidney, and spleen of European domestic ferrets (Mustela putorius furo) by isotope dilution mass spectrometry (IDMS). Housekeeping proteins were chosen that range in abundance to account for different masses of tissue slices of the same organ. Target peptides used for IDMS quantification were conserved across several of the common animal model systems, including baby hamster kidney, mouse, and ferret. Hemagglutinin, the primary antigen of an influenza vaccine, was added at various concentrations to demonstrate the recovery of low-abundance proteins from the complex tissue homogenate. By using housekeeping proteins and a preparation protocol that minimizes sample loss, this study shows that IDMS can accurately quantify proteins in mammalian tissues with unmatched sensitivity and specificity.

The impact of the COVID-19 pandemic on pediatric mortality, including measures to ensure continuity of HIV care, is not well described in Kenya. We evaluated causes of death (COD) among decedents under 5 years of age both before and during the COVID-19 pandemic in Kenya. We analyzed Child Health and Mortality Prevention Surveillance (CHAMPS) data collected in February 2018-March 2022. We describe the proportional contribution of specific conditions in the causal chain of death among decedents aged 28 days to 59 months who underwent minimally invasive tissue (MITS) sampling, had an HIV polymerase chain reaction, and a COD determination. We also calculated all-cause and HIV cause-specific mortality rates using data from two health and demographic surveillance system (HDSS) sites in western Kenya. Results were stratified by time periods: February 2018 to February 2020, and March 2020 to March 2022. Among 269 MITS-eligible decedents, 55.8% died during the pre-COVID period. Of these, 53.7% were infants (28 days to 11 months), and 9.7% were HIV-positive. Leading causes of death for infants included malnutrition (20.5%), pneumonia (17.5%), sepsis (17.1%), and malaria (14.5%). For older children (12-59 months), the predominant causes were malaria (25.6%), malnutrition (21.1%), pneumonia (14.1%), and sepsis (13.1%). All-cause mortality rates did not differ significantly between the periods (53.9 vs. 52.8 per 1,000 live births, p=0.77), but HIV cause-specific mortality rates were significantly lower during March 2020-March 2022 compared to February 2018-February 2020 (1.2 vs. 3.1 per 1,000 live births, p=0.01). Malaria, malnutrition, pneumonia, and sepsis were the leading COD among decedents aged 28 days to 59 months enrolled in CHAMPS between February 2018 and March 2022. These findings may point to the need for urgent, focused efforts to prevent avoidable child deaths. Continued monitoring of HIV-related mortality could provide insights into the ongoing impact of the HIV program in the region.

BACKGROUND: There is paucity of information on the role of cytomegalovirus (CMV) infection as a cause of stillbirths or childhood deaths in low-and middle-income countries (LMICs). We investigated attribution of CMV-disease in the causal pathway to stillbirths and deaths in children <5 years of age in seven LMICs participating in the Child Health and Mortality Prevention Surveillance (CHAMPS) network. METHODS: We analyzed stillbirths and decedents enrolled between December 2016 and July 2023. Deaths were investigated using post-mortem minimally invasive tissue sampling with histopathology and molecular diagnostic investigations of tissues and body fluids, along with review of clinical records. Multi-disciplinary expert panels reviewed findings and reported on the causal pathway to death. RESULTS: CMV was detected in 19.5%(1140/5841) of all evaluated deaths, including 5.0% (111/2204), 6.2% (139/2229), 41.2% (107/260), 68.1%(323/474) and 68.2%(460/674) of stillbirths, neonates (deaths 0-<28 days postnatal), young infants (28-<90 days), older infants (90-<365 days) and children (12-<60 months), respectively. CMV-disease was attributed in the causal pathway to death in 0.9%(20/2204) of stillbirths, 0.8%(17/2229) of neonates, 13.1% (34/260) of young infants, 9.7%(46/474) of older infants and 3.3%(22/674) of children. Decedents with CMV-disease compared with those without CMV-disease in the causal pathway, were more likely to have severe microcephaly (38.2% vs. 21.1%; aOR 2.2, 95%CI: 1.3-3.6) and HIV-infected (36.9% vs. 6.2%; aOR: 10.9, 95%CI: 6.5-18.5). CONCLUSIONS: CMV-disease is an important contributor to deaths during infancy and childhood and is often associated with severe microcephaly and HIV-infection. Improving management of CMV in HIV-infected children and a vaccine to prevent CMV are needed interventions.

Vancomycin-resistant Staphylococcus aureus (VRSA) is a rare but serious public health concern. We describe a VRSA case in North Carolina, USA. The isolate from the case belonged to the USA600 lineage and clonal complex 45. No transmission was identified. Confirmed VRSA cases should include a thorough investigation and public health response.

BACKGROUND: Feedback reports summarizing clinician performance are effective tools for improving antibiotic use in the ambulatory setting, but the effectiveness of feedback reports in the hospital setting is unknown. METHODS: Quasi-experimental study conducted between December 2021 and November 2023 within a pediatric health system measuring the impact of clinician feedback reports delivered by email and reviewed in a monthly meeting on appropriate antibiotic use in children hospitalized with community-acquired pneumonia (CAP). We used an interrupted time series analysis (ITSA) to estimate the immediate change and change over time in the proportion of CAP encounters adherent to validated metrics of antibiotic choice and duration, then used Poisson regression to estimate intervention effect as a rate ratio (RR). RESULTS: Preintervention, 213 of 413 (52%) encounters received the appropriate antibiotic choice and duration, which increased to 308 of 387 (80%) postintervention. The ITSA demonstrated an immediate 18% increase in the proportion of CAP encounters receiving both the appropriate antibiotic choice and duration (95% confidence interval, 3-33%), with no further change over time (-0.3% per month, 95% CI -2-2%). In the Poisson model adjusted for age, sex, race, season, site, and intensive care unit admission, the intervention was associated with a 32% increase in the rate of appropriate antibiotic choice and duration (RR 1.32, 95% confidence interval 1.12-1.56, P <0.01). No difference in length of stay or revisits were detected postintervention. CONCLUSION: The intervention was associated with an increase in clinician adherence to antibiotic choice and duration recommendations for children hospitalized with CAP.

Influenza viruses continually evolve new antigenic variants, through mutations in epitopes of their major surface proteins, hemagglutinin (HA) and neuraminidase (NA). Antigenic drift potentiates the reinfection of previously infected individuals, but the contribution of this process to variability in annual epidemics is not well understood. Here, we link influenza A(H3N2) virus evolution to regional epidemic dynamics in the United States during 1997-2019. We integrate phenotypic measures of HA antigenic drift and sequence-based measures of HA and NA fitness to infer antigenic and genetic distances between viruses circulating in successive seasons. We estimate the magnitude, severity, timing, transmission rate, age-specific patterns, and subtype dominance of each regional outbreak and find that genetic distance based on broad sets of epitope sites is the strongest evolutionary predictor of A(H3N2) virus epidemiology. Increased HA and NA epitope distance between seasons correlates with larger, more intense epidemics, higher transmission, greater A(H3N2) subtype dominance, and a greater proportion of cases in adults relative to children, consistent with increased population susceptibility. Based on random forest models, A(H1N1) incidence impacts A(H3N2) epidemics to a greater extent than viral evolution, suggesting that subtype interference is a major driver of influenza A virus infection ynamics, presumably via heterosubtypic cross-immunity. | Seasonal influenza (flu) viruses cause outbreaks every winter. People infected with influenza typically develop mild respiratory symptoms. But flu infections can cause serious illness in young children, older adults and people with chronic medical conditions. Infected or vaccinated individuals develop some immunity, but the viruses evolve quickly to evade these defenses in a process called antigenic drift. As the viruses change, they can re-infect previously immune people. Scientists update the flu vaccine yearly to keep up with this antigenic drift. The immune system fights flu infections by recognizing two proteins, known as antigens, on the virus’s surface, called hemagglutinin (HA) and neuraminidase (NA). However, mutations in the genes encoding these proteins can make them unrecognizable, letting the virus slip past the immune system. Scientists would like to know how these changes affect the size, severity and timing of annual influenza outbreaks. Perofsky et al. show that tracking genetic changes in HA and NA may help improve flu season predictions. The experiments compared the severity of 22 flu seasons caused by the A(H3N2) subtype in the United States with how much HA and NA had evolved since the previous year. The A(H3N2) subtype experiences the fastest rates of antigenic drift and causes more cases and deaths than other seasonal flu viruses. Genetic changes in HA and NA were a better predictor of A(H3N2) outbreak severity than the blood tests for protective antibodies that epidemiologists traditionally use to track flu evolution. However, the prevalence of another subtype of influenza A circulating in the population, called A(H1N1), was an even better predictor of how severe A(H3N2) outbreaks would be. Perofsky et al. are the first to show that genetic changes in NA contribute to the severity of flu seasons. Previous studies suggested a link between genetic changes in HA and flu season severity, and flu vaccines include the HA protein to help the body recognize new influenza strains. The results suggest that adding the NA protein to flu vaccines may improve their effectiveness. In the future, flu forecasters may want to analyze genetic changes in both NA and HA to make their outbreak predictions. Tracking how much of the A(H1N1) subtype is circulating may also be useful for predicting the severity of A(H3N2) outbreaks. | eng

BACKGROUND: Child mortality in Kenya is 41 per 1,000 live births, despite extensive investment in maternal, newborn, and child health interventions. Caregivers' health-seeking for childhood illness is an important determinant of child survival, and delayed healthcare is associated with high child mortality. We explore determinants of health-seeking decisions for childhood illnesses among caregivers in western Kenya. METHODS: We conducted a qualitative study of 88 community members between April 2017 and February 2018 using purposive sampling in an informal urban settlement in Kisumu County, and in rural Siaya County. Key informant interviews, semi-structured interviews and focus group discussions were performed. We adopted the Partners for Applied Social Sciences model focusing on factors that influence the decision-making process to seek healthcare for sick infants and children. The discussions were audio-recorded and transcribed. Data management was completed on Nvivo® software. Iterative analysis process was utilized and themes were identified and collated. RESULTS: Our findings reveal four thematic areas: Illness interpretation, the role of social relationship on illness recognition and response, medical pluralism and healthcare access. Participants reported some illnesses are caused by supernatural powers and some by biological factors, and that the illness etiology would determine the health-seeking pathway. It was common to seek consensus from respected community members on the diagnosis and therefore presumed cause and necessary treatment for a child's illness. Medical pluralism was commonly practiced and caregivers would alternate between biomedicine and traditional medicine. Accessibility of healthcare may determine the health seeking pathway. Caregivers unable to afford biomedical care may choose traditional medicine as a cheaper alternative. CONCLUSION: Health seeking behavior was driven by illness interpretation, financial cost associated with healthcare and advice from extended family and community. These findings enrich the perspectives of health education programs to develop health messages that address factors that hinder prompt health care seeking.

IMPORTANCE: The emergence of acute neurological symptoms in children necessitates immediate intervention. Although low- and middle-income countries (LMICs) bear the highest burden of neurological diseases, there is a scarcity of diagnostic and therapeutic resources. Therefore, current understanding of the etiology of neurological emergencies in LMICs relies mainly on clinical diagnoses and verbal autopsies. OBJECTIVE: To characterize the association of premortem neurological symptoms and their management with postmortem-confirmed cause of death among children aged younger than 5 years in LMICs and to identify current gaps and improve strategies to enhance child survival. DESIGN, SETTING, AND PARTICIPANTS: This cross-sectional study was conducted between December 3, 2016, and July 22, 2022, at the 7 participating sites in the Child Health and Mortality Prevention Surveillance (CHAMPS) network (Bangladesh, Ethiopia, Kenya, Mali, Mozambique, Sierra Leone, and South Africa). Minimally invasive tissue sampling was performed at the CHAMPS sites with specimens from deceased children aged younger than 5 years. This study included deceased children who underwent a premortem neurological evaluation and had a postmortem-confirmed cause of death. Data analysis was performed between July 22, 2022, and January 15, 2023. MAIN OUTCOMES AND MEASURES: Descriptive analysis was performed using neurological evaluations from premortem clinical records and from postmortem determination of cause of death (based on histopathology, microbiological testing, clinical records, and verbal autopsies). RESULTS: Of the 2127 deaths of children codified during the study period, 1330 (62.5%) had neurological evaluations recorded and were included in this analysis. The 1330 children had a median age of 11 (IQR, 2-324) days; 745 (56.0%) were male and 727 (54.7%) presented with neurological symptoms during illness before death. The most common postmortem-confirmed neurological diagnoses related to death were hypoxic events (308 [23.2%]), meningoencephalitis (135 [10.2%]), and cerebral malaria (68 [5.1%]). There were 12 neonates with overlapping hypoxic events and meningoencephalitis, but there were no patients with overlapping meningoencephalitis and cerebral malaria. Neurological symptoms were similar among diagnoses, and no combination of symptoms was accurate in differentiating them without complementary tools. However, only 25 children (18.5%) with meningitis had a lumbar puncture performed before death. Nearly 90% of deaths (442 of 511 [86.5%]) with neurological diagnoses in the chain of events leading to death were considered preventable. CONCLUSIONS AND RELEVANCE: In this cross-sectional study of children aged younger than 5 years, neurological symptoms were frequent before death. However, clinical phenotypes were insufficient to differentiate the most common underlying neurological diagnoses. The low rate of lumbar punctures performed was especially worrying, suggesting a challenge in quality of care of children presenting with neurological symptoms. Improved diagnostic management of neurological emergencies is necessary to ultimately reduce mortality in this vulnerable population.

Wisdom is the hallmark of social judgment, but how people across cultures recognize wisdom remains unclear-distinct philosophical traditions suggest different views of wisdom's cardinal features. We explore perception of wise minds across 16 socio-economically and culturally diverse convenience samples from 12 countries. Participants assessed wisdom exemplars, non-exemplars, and themselves on 19 socio-cognitive characteristics, subsequently rating targets' wisdom, knowledge, and understanding. Analyses reveal two positively related dimensions-Reflective Orientation and Socio-Emotional Awareness. These dimensions are consistent across the studied cultural regions and interact when informing wisdom ratings: wisest targets-as perceived by participants-score high on both dimensions, whereas the least wise are not reflective but moderately socio-emotional. Additionally, individuals view themselves as less reflective but more socio-emotionally aware than most wisdom exemplars. Our findings expand folk psychology and social judgment research beyond the Global North, showing how individuals perceive desirable cognitive and socio-emotional qualities, and contribute to an understanding of mind perception.

Abrin and ricin are toxic proteins produced by plants. Both proteins are composed of two subunits, an A-chain and a B-chain. The A-chain is responsible for the enzymatic activity, which causes toxicity. The B-chain binds to glycoproteins on the cell surface to direct the A-chain to its target. Both toxins depurinate 28S rRNA, making it impossible to differentiate these toxins based on only their enzymatic activity. We developed an analytical workflow for both ricin and abrin using a single method and sample. We have developed a novel affinity enrichment technique based on the ability of the B-chain to bind a glycoprotein, asialofetuin. After the toxin is extracted with asialofetuin-coated magnetic beads, an RNA substrate is added. Then, depurination is detected by a benchtop matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometer to determine the presence or absence of an active toxin. Next, the beads are subjected to tryptic digest. Toxin fingerprinting is done on a benchtop MALDI-TOF MS. We validated the assay through sensitivity and specificity studies and determined the limit of detection for each toxin as nanogram level for enzymatic activity and μg level for toxin fingerprinting. We examined potential cross-reactivity from proteins that are near neighbors of the toxins and examined potential false results in the presence of white powders.

SARS-CoV-2 spike (S) proteins undergo extensive glycosylation, aiding in proper folding, enhancing stability, and evading host immune surveillance. In this study, we used mass spectrometric analysis to elucidate the N-glycosylation characteristics and disulfide bonding of recombinant spike proteins derived from the SARS-CoV-2 Omicron variant (B.1.1.529) in comparison with the D614G spike variant. Furthermore, we conducted microsecond-long molecular dynamics simulations on spike proteins to resolve how the different N-glycans impact spike conformational sampling in the two variants. Our findings reveal that the Omicron spike protein maintains an overall resemblance to the D614G spike variant in terms of site-specific glycan processing and disulfide bond formation. Nonetheless, alterations in glycans were observed at certain N-glycosylation sites. These changes, in synergy with mutations within the Omicron spike protein, result in increased surface accessibility of the macromolecule, including the ectodomain, receptor-binding domain, and N-terminal domain. Additionally, mutagenesis and pull-down assays reveal the role of glycosylation of a specific sequon (N149); furthermore, the correlation of MD simulation and HDX-MS identified several high-dynamic areas of the spike proteins. These insights contribute to our understanding of the interplay between structure and function, thereby advancing effective vaccination and therapeutic strategies.

BACKGROUND: Annual influenza vaccination is recommended for older adults but repeated vaccination with standard-dose influenza vaccine has been linked to reduced immunogenicity and effectiveness, especially against A(H3N2) viruses. METHODS: Community-dwelling Hong Kong adults aged 65-82 years were randomly allocated to receive 2017-2018 standard-dose quadrivalent, MF59-adjuvanted trivalent, high-dose trivalent, and recombinant-HA quadrivalent vaccination. Antibody response to unchanged A(H3N2) vaccine antigen was compared among participants with and without self-reported prior year (2016-2017) standard-dose vaccination. RESULTS: Mean fold rise (MFR) in antibody titers from day 0 to day 30 by hemagglutination inhibition and virus microneutralization assays were lower among 2017-2018 standard-dose and enhanced vaccine recipients with (range, 1.7-3.0) versus without (range, 4.3-14.3) prior 2016-2017 vaccination. MFR was significantly reduced by about one-half to four-fifths for previously vaccinated recipients of standard-dose and all 3 enhanced vaccines (β range, .21-.48). Among prior-year vaccinated older adults, enhanced vaccines induced higher 1.43 to 2.39-fold geometric mean titers and 1.28 to 1.74-fold MFR versus standard-dose vaccine by microneutralization assay. CONCLUSIONS: In the context of unchanged A(H3N2) vaccine strain, prior-year vaccination was associated with reduced antibody response among both standard-dose and enhanced influenza vaccine recipients. Enhanced vaccines improved antibody response among older adults with prior-year standard-dose vaccination.

Botulism is a paralytic disease due to the inhibition of acetylcholine exocytosis at the neuromuscular junction, which can be lethal if left untreated. Botulinum neurotoxins (BoNTs) are produced by some spore-forming Clostridium bacteria. The current confirmatory assay to test for BoNTs in clinical specimens is the gold-standard mouse bioassay. However, an Endopep-MS assay method has been developed to detect BoNTs in clinical samples using benchtop mass spectrometric detection. This work demonstrates the validation of the Endopep-MS method for clinical specimens with the intent of method distribution in public health laboratories. The Endopep-MS assay was validated by assessing the sensitivity, robustness, selectivity, specificity, and reproducibility. The limit of detection was found to be equivalent to or more sensitive than the mouse bioassay. Specificity studies determined no cross-reactivity between the different serotypes and no false positives from an exclusivity panel of culture supernatants of enteric disease organisms and non-toxigenic strains of Clostridium. Inter-serotype specificity testing with 19 BoNT subtypes was 100% concordant with the expected results, accurately determining the presence of the correct serotype and the absence of incorrect serotypes. Additionally, a panel of potential interfering substances was used to test selectivity. Finally, clinical studies included clinical specimen stability and reproducibility, which was found to be 99.9% from a multicenter evaluation study. The multicenter validation study also included a clinical validation study, which yielded a 99.4% correct determination rate. Use of the Endopep-MS method will improve the capacity and response time for laboratory confirmation of botulism in public health laboratories.

BACKGROUND: Lipid profiling is central for coronary artery disease (CAD) risk assessment. Nonadherence or unreported use of lipid-lowering drugs, particularly statins, can significantly complicate the association between lipid profile measures and CAD clinical outcomes. By combining medication history evaluation with statin analysis in plasma, we determined the effects of inaccurately reported statin use on lipid profile measures and their association with CAD risk. METHODS: We compared medication history of statin use with statin concentration measurements, by liquid chromatography-tandem mass spectrometry, in 690 participants undergoing coronary angiography (63 ± 11 years of age). Nominal logistic regression was employed to model CAD diagnosis with statin measurements, phenotypic, and lipid profile characteristics. RESULTS: Medication history of statin use was confirmed by statin assay for 81% of the patients. Surprisingly, statins were detected in 46% of patients without statin use records. Nonreported statin use was disproportionately higher among older participants. Stratifying samples by statin history resulted in underestimated LDL-lipid measures. Apolipoprotein B concentrations had a significant inverse CAD association, which became nonsignificant upon re-stratification using the statin assay data. CONCLUSIONS: Our study uncovered prominent discrepancies between medication records and actual statin use measured by mass spectrometry. We showed that inaccurate statin use assessments may lead to overestimation and underestimation of LDL levels in statin user and nonuser categories, exaggerating the reverse epidemiology association between LDL levels and CAD diagnosis. Combining medication history and quantitative statin assay data can significantly improve the design, analysis, and interpretation of clinical and epidemiological studies.

Delays in illness recognition, healthcare seeking, and in the provision of appropriate clinical care are common in resource-limited settings. Our objective was to determine the frequency of delays in the "Three Delays-in-Healthcare", and factors associated with delays, among deceased infants and children in seven countries with high childhood mortality. We conducted a retrospective, descriptive study using data from verbal autopsies and medical records for infants and children aged 1-59 months who died between December 2016 and February 2022 in six sites in sub-Saharan Africa and one in South Asia (Bangladesh) and were enrolled in Child Health and Mortality Prevention Surveillance (CHAMPS). Delays in 1) illness recognition in the home/decision to seek care, 2) transportation to healthcare facilities, and 3) the receipt of clinical care in healthcare facilities were categorized according to the "Three Delays-in-Healthcare". Comparisons in factors associated with delays were made using Chi-square testing. Information was available for 1,326 deaths among infants and under 5 children. The majority had at least one identified delay (n = 854, 64%). Waiting >72 hours after illness recognition to seek health care (n = 422, 32%) was the most common delay. Challenges in obtaining transportation occurred infrequently when seeking care (n = 51, 4%). In healthcare facilities, prescribed medications were sometimes unavailable (n = 102, 8%). Deceased children aged 12-59 months experienced more delay than infants aged 1-11 months (68% vs. 61%, P = 0.018). Delays in seeking clinical care were common among deceased infants and children. Additional study to assess the frequency of delays in seeking clinical care and its provision among children who survive is warranted.

BACKGROUND: Malaria is a leading cause of childhood mortality worldwide. However, accurate estimates of malaria prevalence and causality among patients who die at the country level are lacking due to the limited specificity of diagnostic tools used to attribute etiologies. Accurate estimates are crucial for prioritizing interventions and resources aimed at reducing malaria-related mortality. METHODS: Seven Child Health and Mortality Prevention Surveillance (CHAMPS) Network sites collected comprehensive data on stillbirths and children <5 years, using minimally invasive tissue sampling (MITS). A DeCoDe (Determination of Cause of Death) panel employed standardized protocols for assigning underlying, intermediate, and immediate causes of death, integrating sociodemographic, clinical, laboratory (including extensive microbiology, histopathology, and malaria testing), and verbal autopsy data. Analyses were conducted to ascertain the strength of evidence for cause of death (CoD), describe factors associated with malaria-related deaths, estimate malaria-specific mortality, and assess the proportion of preventable deaths. FINDINGS: Between December 3, 2016, and December 31, 2022, 2673 deaths underwent MITS and had a CoD attributed from four CHAMPS sites with at least 1 malaria-attributed death. No malaria-attributable deaths were documented among 891 stillbirths or 924 neonatal deaths, therefore this analysis concentrates on the remaining 858 deaths among children aged 1-59 months. Malaria was in the causal chain for 42.9% (126/294) of deaths from Sierra Leone, 31.4% (96/306) in Kenya, 18.2% (36/198) in Mozambique, 6.7% (4/60) in Mali, and 0.3% (1/292) in South Africa. Compared to non-malaria related deaths, malaria-related deaths skewed towards older infants and children (p<0.001), with 71.0% among ages 12-59 months. Malaria was the sole infecting pathogen in 184 (70.2%) of malaria-attributed deaths, whereas bacterial and viral co-infections were identified in the causal pathway in 24·0% and 12.2% of cases, respectively. Malnutrition was found at a similar level in the causal pathway of both malaria (26.7%) and non-malaria (30.7%, p=0.256) deaths. Less than two-thirds (164/262; 62.6%) of malaria deaths had received antimalarials prior to death. Nearly all (98·9%) malaria-related deaths were deemed preventable. INTERPRETATION: Malaria remains a significant cause of childhood mortality in the CHAMPS malaria-endemic sites. The high bacterial co-infection prevalence among malaria deaths underscores the potential benefits of antibiotics for severe malaria patients. Compared to non-malaria deaths, many of malaria-attributed deaths are preventable through accessible malaria control measures. FUNDING: This work was supported by the Bill & Melinda Gates Foundation [OPP1126780].

Rationale: Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat-denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. Methods: We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions; if high-energy collision-induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low-energy collision-induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX-MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2O. Results: We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements. Conclusion: Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors. Published 2024. This article is a U.S. Government work and is in the public domain in the USA.

BACKGROUND: Humans are likely exposed to microplastics (MPs) in a variety of places including indoor and outdoor air. Research to better understand how exposure to MPs correlates to health is growing. To fully understand the possible impacts of MPs on human health, it is necessary to quantify MP exposure and identify what critical data gaps exist. OBJECTIVES: The current paper provides a human exposure assessment of microplastics in the air using systematically reviewed literature that provided concentration of MPs in air as well as doses used in toxicology studies to calculate inhalation exposure dose. METHODS: All published peer-reviewed journal articles, non-published papers, and grey literature that focused on micro- or nano-plastics in indoor and outdoor air were systematically searched using PRISMA guidelines. Literature that defined specific concentrations and size of MPs in air or exposed to human lung cells, animals, or humans with measurable health impacts were included in data extraction. Inhalational exposures were calculated for different age groups using published MP concentrations from the included literature using exposure dose equations and values from U.S. ATSDR and EPA. RESULTS: Calculated mean indoor inhalational exposures from passive sampling methods were higher than those calculated from active sampling methods. When comparing indoor and outdoor sampling, calculated inhalation exposures from indoor samples were greater than those from outdoor samples. Inhalation exposures of MPs differed between age groups with infants having the highest calculated dose values for all locations followed by preschool age children, middle-school aged children, pregnant women, adolescents, and non-pregnant adults. MP doses used in toxicology studies produced higher calculated mean inhalational exposures than those from environmental samples. IMPACT: This study is the first known systematic review of inhalational MP exposure from indoor and outdoor air. It also provides inhalational exposures calculated from previously published environmental samples of MPs as well as from toxicology studies.

This manuscript describes the use of an analytical assay that combines transfection of mammalian cells and isotope dilution mass spectrometry (IDMS) for accurate quantification of antigen expression. Expired mRNA COVID-19 vaccine material was stored at 4 °C, room temperature (∼25 °C), and 56 °C over a period of 5 weeks. The same vaccine was also exposed to 5 freeze-thaw cycles. Every week, the spike protein antigenic expression in mammalian (BHK-21) cells was evaluated. Housekeeping proteins, β-actin and GAPDH, were simultaneously quantified to account for the variation in cell counts that occurs during maintenance and growth of cell cultures. Data show that vaccine stored at elevated temperatures results in reduced spike protein expression. Also, maintaining the vaccine in ultracold conditions or exposing the vaccine to freeze-thaw cycles had less effect on the vaccine's ability to produce the antigen in mammalian cells. We describe the use of IDMS as an antibody-free means to accurately quantify expressed protein from mammalian cells transfected with mRNA vaccine.

Farmed mink are one of few animals in which infection with SARS-CoV-2 has resulted in sustained transmission among a population and spillback from mink to people. In September 2020, mink on a Michigan farm exhibited increased morbidity and mortality rates due to confirmed SARS-CoV-2 infection. We conducted an epidemiologic investigation to identify the source of initial mink exposure, assess the degree of spread within the facility's overall mink population, and evaluate the risk of further viral spread on the farm and in surrounding wildlife habitats. Three farm employees reported symptoms consistent with COVID-19 the same day that increased mortality rates were observed among the mink herd. One of these individuals, and another asymptomatic employee, tested positive for SARS-CoV-2 by real-time reverse transcription PCR (RT-qPCR) 9 days later. All but one mink sampled on the farm were positive for SARS-CoV-2 based on nucleic acid detection from at least one oral, nasal, or rectal swab tested by RT-qPCR (99%). Sequence analysis showed high degrees of similarity between sequences from mink and the two positive farm employees. Epidemiologic and genomic data, including the presence of F486L and N501T mutations believed to arise through mink adaptation, support the hypothesis that the two employees with SARS-CoV-2 nucleic acid detection contracted COVID-19 from mink. However, the specific source of virus introduction onto the farm was not identified. Three companion animals living with mink farm employees and 31 wild animals of six species sampled in the surrounding area were negative for SARS-CoV-2 by RT-qPCR. Results from this investigation support the necessity of a One Health approach to manage the zoonotic spread of SARS-CoV-2 and underscores the critical need for multifaceted public health approaches to prevent the introduction and spread of respiratory viruses on mink farms.

Objectives. To describe RDS in neonatal deaths at the CHAMPS-Kenya site between 2017 and 2021. Methods. We included 165 neonatal deaths whose their Causes of death (COD) were determined by a panel of experts using data from post-mortem conducted through minimally invasive tissue specimen testing, clinical records, and verbal autopsy. Results. Twenty-six percent (43/165) of neonatal deaths were attributable to RDS. Most cases occurred in low birthweight and preterm neonates. From these cases, less than half of the hospitalizations were diagnosed with RDS before death, and essential diagnostic tests were not performed in most cases. Most cases received suboptimal levels of supplemental oxygen, and critical interventions like surfactant replacement therapy and mechanical ventilation were not adequately utilized when available. Conclusion. The study highlights the urgent need for improved diagnosis and management of RDS, emphasizing the importance of increasing clinical suspicion and enhancing training in its clinical management to reduce mortality rates.

Introduction. Lack of laboratory capacity hampers consistent national antimicrobial resistance (AMR) surveillance. Chromogenic media may provide a practical screening tool for detection of individuals colonized by extended-spectrum beta-lactamase (ESBL)-producing organisms.Hypothesis. CHROMagar ESBL media represent an adequate screening method for the detection of extended-spectrum cephalosporin-resistant Enterobacterales (ESCrE), isolated from rectal swabs.Aim. To evaluate the performance of CHROMagar ESBL media to accurately identify ESCrE isolates from rectal swab samples attained from hospitalized and community participants.Methodology. All participants provided informed consent prior to enrolment. Rectal swabs from 2469 hospital and community participants were inoculated onto CHROMagar ESBL. The performance of CHROMagar ESBL to differentiate Escherichia coli and Klebsiella spp., Enterobacter spp. and Citrobacter spp. (KEC spp.) as well as select for extended-spectrum cephalosporin resistance were compared to matrix-assisted laser desorption/ionization-time-of-flight MS (MALDI-TOF-MS) and VITEK-2 automated susceptibility testing.Results. CHROMagar ESBL had a positive and negative agreement of 91.2 % (95 % CI, 88.4-93.3) and 86.8 % (95 % CI, 82.0-90.7) for E. coli and 88.1 % (95 % CI 83.2-92.1) and 87.6 % (95 % CI 84.7-90.2) for KEC spp. differentiation, respectively, when compared to species ID by MALDI-TOF-MS. When evaluated for phenotypic susceptibilities (VITEK-2), 88.1 % (714/810) of the isolates recovered on the selective agar exhibited resistance to third-generation cephalosporins.Conclusion. The performance characteristics of CHROMagar ESBL media suggest that they may be a viable screening tool for the identification of ESCrE from hospitalized and community participants and could be used to inform infection prevention and control practices in Botswana and potentially other low-and middle-income countries (LMICs). Further studies are required to analyse the costs and the impact on time-to-result of the media in comparison with available laboratory methods for ESCrE surveillance in the country.

Worldwide, nearly six million children under the age of five (<5s) die annually, a substantial proportion of which are due to preventable and treatable diseases. Efforts to reduce child mortality indicators in the most affected regions are often undermined by a lack of accurate cause of death data. To generate timely and more accurate causes of death data for <5s, the Child Health and Mortality Prevention Surveillance (CHAMPS) Network established mortality surveillance in multiple countries using Minimally Invasive Tissue Sampling (MITS) in <5 deaths. Here we present acceptability of MITS by community members and healthcare workers in Siaya and Kisumu counties, western Kenya. From April 2017 to February 2018, we conducted 40 in-depth interviews and five focus group discussions with healthcare workers and community members, before and during CHAMPS implementation. Participants were purposively selected. Field observations to understand traditional death-related practices were also performed. Interviews were transcribed into Nvivo 11.0 for data organization and management. Analysis was guided by the grounded theory approach. Facilitators of acceptability were desire to understand why death occurred, timely performance of MITS procedures, potential for MITS results in improving clinical practice and specific assistance provided to families by the CHAMPS program. However, cultural and religious beliefs highlighted important challenges to acceptability, including CHAMPS teams recruiting after a child's death, rumours and myths, unmet expectations from families, and fear by healthcare workers that some families could use MITS results to sue for negligence. Increasing MITS uptake requires sustained strategies to strengthen the identified facilitators of acceptability and simultaneously address the barriers. MITS acceptance will contribute to better characterization of causes of death and support the development of improved interventions aimed at reducing <5 mortality.

BACKGROUND: Most childhood deaths globally are considered preventable through high-quality clinical care, which includes adherence to clinical care recommendations. Our objective was to describe adherence to World Health Organization recommendations for the management of leading causes of death among children. METHODS: We conducted a retrospective, descriptive study examining clinical data for children aged 1-59 months who were hospitalized and died in a Child Health and Mortality Prevention Surveillance (CHAMPS) catchment, December 2016-June 2021. Catchment areas included: Baliakandi and Faridpur, Bangladesh; Kersa, Haramaya, and Harar, Ethiopia; Kisumu and Siaya, Kenya; Bamako, Mali; Manhiça and Quelimane, Mozambique; Makeni, Sierra Leone; Soweto, South Africa. We reviewed medical records of those who died from lower respiratory tract infections, sepsis, malnutrition, malaria, and diarrheal diseases to determine the proportion who received recommended treatments and compared adherence by hospitalization duration. FINDINGS: CHAMPS enrolled 460 hospitalized children who died from the leading causes (median age 12 months, 53.0% male). Median hospital admission was 31 h. There were 51.0% (n = 127/249) of children who died from lower respiratory tract infections received supplemental oxygen. Administration of intravenous fluids for sepsis (15.9%, n = 36/226) and supplemental feeds for malnutrition (14.0%, n = 18/129) were uncommon. There were 51.4% (n = 55/107) of those who died from malaria received antimalarials. Of the 80 children who died from diarrheal diseases, 76.2% received intravenous fluids. Those admitted for ≥24 h more commonly received antibiotics for lower respiratory tract infections and sepsis, supplemental feeds for malnutrition, and intravenous fluids for sepsis than those admitted <24 h. INTERPRETATION: Provision of recommended clinical care for leading causes of death among young children was suboptimal. Further studies are needed to understand the reasons for deficits in clinical care recommendation adherence. FUNDING: Bill & Melinda Gates Foundation.

BACKGROUND: Invasive Group B Streptococcus (GBS) is a common cause of early-onset neonatal sepsis and is also associated with stillbirth. This study aimed to determine the proportion of stillborn infants and infants who died between 0 and 90 days attributable to GBS using postmortem minimally invasive tissue sampling (MITS) in 7 low- and middle-income countries (LMICs) participating in Child Health and Mortality Prevention Surveillance (CHAMPS). METHODS: Deaths that occurred between December 2016 and December 2021 were investigated with MITS, including culture for bacteria of blood and cerebrospinal fluid (CSF), multipathogen polymerase chain reaction on blood, CSF, and lung tissue and histopathology of lung, liver, and brain. Data collection included clinical record review and verbal autopsy. Expert panels reviewed all information and assigned causes of death. RESULTS: We evaluated 2966 deaths, including stillborn infants (n = 1322), infants who died during first day of life (0 to <24 hours, n = 597), early neonatal deaths (END) (1 day to <7 days; END; n = 593), and deaths from 7 to 90 days (n = 454). Group B Streptococcus was determined to be in the causal pathway of death for 2.7% of infants (79 of 2, 966; range, 0.3% in Sierra Leone to 7.2% in South Africa), including 2.3% (31 of 1322) of stillbirths, 4.7% (28 of 597) 0 to <24 hours, 1.9% (11 of 593) END, and 2.0% (9 of 454) of deaths from 7 to 90 days of age. Among deaths attributed to GBS with birth weight data available, 61.9% (39 of 63) of decedents weighed <2500 grams at birth. Group B Streptococcus sepsis was the postmortem diagnosis for 100% (31 of 31) of stillbirths. For deaths <90 days, postmortem diagnoses included GBS sepsis (83.3%, 40 of 48), GBS meningitis (4.2%, 2 of 48), and GBS pneumonia (2.1%, 1 of 48). CONCLUSIONS: Our study reveals significant heterogeneity in the contribution of invasive GBS disease to infant mortality across different countries, emphasizing the need for tailored prevention strategies. Moreover, our findings highlight the substantial impact of GBS on stillbirths, shedding light on a previously underestimated aspect in LMICs.

INTRODUCTION: Postpartum hemorrhage (PPH) is a significant cause of maternal mortality worldwide, particularly in low- and middle-income countries. It is essential to develop effective prediction models to identify women at risk of PPH and implement appropriate interventions to reduce maternal morbidity and mortality. This study aims to predict the occurrence of postpartum hemorrhage using machine learning models based on antenatal, intrapartum, and postnatal visit data obtained from the Kenya Antenatal and Postnatal Care Research Collective cohort. METHOD: Four machine learning models - logistic regression, naïve Bayes, decision tree, and random forest - were constructed using 67% training data (1,056/1,576). The training data was further split into 67% for model building and 33% cross validation. Once the models are built, the remaining 33% (520/1,576) independent test data was used for external validation to confirm the models' performance. Models were fine-tuned using feature selection through extra tree classifier technique. Model performance was assessed using accuracy, sensitivity, and area under the curve (AUC) of the receiver operating characteristics (ROC) curve. RESULT: The naïve Bayes model performed best with 0.95 accuracy, 0.97 specificity, and 0.76 AUC. Seven factors (anemia, limited prenatal care, hemoglobin concentrations, signs of pallor at intrapartum, intrapartum systolic blood pressure, intrapartum diastolic blood pressure, and intrapartum respiratory rate) were associated with PPH prediction in Kenyan population. DISCUSSION: This study demonstrates the potential of machine learning models in predicting PPH in the Kenyan population. Future studies with larger datasets and more PPH cases should be conducted to improve prediction performance of machine learning model. Such prediction algorithms would immensely help to construct a personalized obstetric path for each pregnant patient, improve resource allocation, and reduce maternal mortality and morbidity.