Single-Molecule Real-Time (SMRT) Full-Length RNA-Sequencing Reveals Novel and Distinct mRNA Isoforms in Human Bone Marrow Cell Subpopulations.

Hematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single-molecule real-time (SMRT) full-length RNA-sequencing. This analysis revealed a ~5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of messenger RNA (mRNA) isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was evaluated by mass spectrometry and validated previously unknown protein isoforms predicted e.g., for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g., CFD, GATA2, HLA-A, B, and C) also distinguished cell subpopulations but were only detectable by full-length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis

A pooled testing strategy for identifying SARS-CoV-2 at low prevalence

Suppressing infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will probably require the rapid identification and isolation of individuals infected with the virus on an ongoing basis. Reverse-transcription polymerase chain reaction (RT-PCR) tests are accurate but costly, which makes the regular testing of every individual expensive. These costs are a challenge for all countries around the world, but particularly for low-to-middle-income countries. Cost reductions can be achieved by pooling (or combining) subsamples and testing them in groups1-7. A balance must be struck between increasing the group size and retaining test sensitivity, as sample dilution increases the likelihood of false-negative test results for individuals with a low viral load in the sampled region at the time of the test8. Similarly, minimizing the number of tests to reduce costs must be balanced against minimizing the time that testing takes, to reduce the spread of the infection. Here we propose an algorithm for pooling subsamples based on the geometry of a hypercube that, at low prevalence, accurately identifies individuals infected with SARS-CoV-2 in a small number of tests and few rounds of testing. We discuss the optimal group size and explain why, given the highly infectious nature of the disease, largely parallel searches are preferred. We report proof-of-concept experiments in which a positive subsample was detected even when diluted 100-fold with negative subsamples (compared with 30-48-fold dilutions described in previous studies9-11). We quantify the loss of sensitivity due to dilution and discuss how it may be mitigated by the frequent re-testing of groups, for example. With the use of these methods, the cost of mass testing could be reduced by a large factor. At low prevalence, the costs decrease in rough proportion to the prevalence. Field trials of our approach are under way in Rwanda and South Africa. The use of group testing on a massive scale to monitor infection rates closely and continually in a population, along with the rapid and effective isolation of people with SARS-CoV-2 infections, provides a promising pathway towards the long-term control of coronavirus disease 2019 (COVID-19).info:eu-repo/semantics/publishe

Effects of antibiotic resistance, drug target attainment, bacterial pathogenicity and virulence, and antibiotic access and affordability on outcomes in neonatal sepsis: an international microbiology and drug evaluation prospective substudy (BARNARDS).

BACKGROUND: Sepsis is a major contributor to neonatal mortality, particularly in low-income and middle-income countries (LMICs). WHO advocates ampicillin-gentamicin as first-line therapy for the management of neonatal sepsis. In the BARNARDS observational cohort study of neonatal sepsis and antimicrobial resistance in LMICs, common sepsis pathogens were characterised via whole genome sequencing (WGS) and antimicrobial resistance profiles. In this substudy of BARNARDS, we aimed to assess the use and efficacy of empirical antibiotic therapies commonly used in LMICs for neonatal sepsis. METHODS: In BARNARDS, consenting mother-neonates aged 0-60 days dyads were enrolled on delivery or neonatal presentation with suspected sepsis at 12 BARNARDS clinical sites in Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Stillborn babies were excluded from the study. Blood samples were collected from neonates presenting with clinical signs of sepsis, and WGS and minimum inhibitory concentrations for antibiotic treatment were determined for bacterial isolates from culture-confirmed sepsis. Neonatal outcome data were collected following enrolment until 60 days of life. Antibiotic usage and neonatal outcome data were assessed. Survival analyses were adjusted to take into account potential clinical confounding variables related to the birth and pathogen. Additionally, resistance profiles, pharmacokinetic-pharmacodynamic probability of target attainment, and frequency of resistance (ie, resistance defined by in-vitro growth of isolates when challenged by antibiotics) were assessed. Questionnaires on health structures and antibiotic costs evaluated accessibility and affordability. FINDINGS: Between Nov 12, 2015, and Feb 1, 2018, 36 285 neonates were enrolled into the main BARNARDS study, of whom 9874 had clinically diagnosed sepsis and 5749 had available antibiotic data. The four most commonly prescribed antibiotic combinations given to 4451 neonates (77·42%) of 5749 were ampicillin-gentamicin, ceftazidime-amikacin, piperacillin-tazobactam-amikacin, and amoxicillin clavulanate-amikacin. This dataset assessed 476 prescriptions for 442 neonates treated with one of these antibiotic combinations with WGS data (all BARNARDS countries were represented in this subset except India). Multiple pathogens were isolated, totalling 457 isolates. Reported mortality was lower for neonates treated with ceftazidime-amikacin than for neonates treated with ampicillin-gentamicin (hazard ratio [adjusted for clinical variables considered potential confounders to outcomes] 0·32, 95% CI 0·14-0·72; p=0·0060). Of 390 Gram-negative isolates, 379 (97·2%) were resistant to ampicillin and 274 (70·3%) were resistant to gentamicin. Susceptibility of Gram-negative isolates to at least one antibiotic in a treatment combination was noted in 111 (28·5%) to ampicillin-gentamicin; 286 (73·3%) to amoxicillin clavulanate-amikacin; 301 (77·2%) to ceftazidime-amikacin; and 312 (80·0%) to piperacillin-tazobactam-amikacin. A probability of target attainment of 80% or more was noted in 26 neonates (33·7% [SD 0·59]) of 78 with ampicillin-gentamicin; 15 (68·0% [3·84]) of 27 with amoxicillin clavulanate-amikacin; 93 (92·7% [0·24]) of 109 with ceftazidime-amikacin; and 70 (85·3% [0·47]) of 76 with piperacillin-tazobactam-amikacin. However, antibiotic and country effects could not be distinguished. Frequency of resistance was recorded most frequently with fosfomycin (in 78 isolates [68·4%] of 114), followed by colistin (55 isolates [57·3%] of 96), and gentamicin (62 isolates [53·0%] of 117). Sites in six of the seven countries (excluding South Africa) stated that the cost of antibiotics would influence treatment of neonatal sepsis. INTERPRETATION: Our data raise questions about the empirical use of combined ampicillin-gentamicin for neonatal sepsis in LMICs because of its high resistance and high rates of frequency of resistance and low probability of target attainment. Accessibility and affordability need to be considered when advocating antibiotic treatments with variance in economic health structures across LMICs. FUNDING: The Bill & Melinda Gates Foundation

Characterization of antimicrobial resistant Gram-negative bacteria that cause neonatal sepsis in seven low and middle-income countries

Antimicrobial resistance in neonatal sepsis is rising, yet mechanisms of resistance that often spread between species via mobile genetic elements, ultimately limiting treatments in low- and middle-income countries (LMICs), are poorly characterized. The Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) network was initiated to characterize the cause and burden of antimicrobial resistance in neonatal sepsis for seven LMICs in Africa and South Asia. A total of 36,285 neonates were enrolled in the BARNARDS study between November 2015 and December 2017, of whom 2,483 were diagnosed with culture-confirmed sepsis. Klebsiella pneumoniae (n = 258) was the main cause of neonatal sepsis, with Serratia marcescens (n = 151), Klebsiella michiganensis (n = 117), Escherichia coli (n = 75) and Enterobacter cloacae complex (n = 57) also detected. We present whole-genome sequencing, antimicrobial susceptibility and clinical data for 916 out of 1,038 neonatal sepsis isolates (97 isolates were not recovered from initial isolation at local sites). Enterobacterales (K. pneumoniae, E. coli and E. cloacae) harboured multiple cephalosporin and carbapenem resistance genes. All isolated pathogens were resistant to multiple antibiotic classes, including those used to treat neonatal sepsis. Intraspecies diversity of K. pneumoniae and E. coli indicated that multiple antibiotic-resistant lineages cause neonatal sepsis. Our results will underpin research towards better treatments for neonatal sepsis in LMICs

Characterisation of Staphylococci species from neonatal blood cultures in low- and middle-income countries

Background: In low- and middle-income countries (LMIC) Staphylococcus aureus is regarded as one of the leading bacterial causes of neonatal sepsis, however there is limited knowledge on the species diversity and antimicrobial resistance caused by Gram-positive bacteria (GPB). Methods: We characterised GPB isolates from neonatal blood cultures from LMICs in Africa (Ethiopia, Nigeria, Rwanda, and South Africa) and South-Asia (Bangladesh and Pakistan) between 2015–2017. We determined minimum inhibitory concentrations and performed whole genome sequencing (WGS) on Staphylococci isolates recovered and clinical data collected related to the onset of sepsis and the outcome of the neonate up to 60 days of age. Results: From the isolates recovered from blood cultures, Staphylococci species were most frequently identified. Out of 100 S. aureus isolates sequenced, 18 different sequence types (ST) were found which unveiled two small epidemiological clusters caused by methicillin resistant S. aureus (MRSA) in Pakistan (ST8) and South Africa (ST5), both with high mortality (n = 6/17). One-third of S. aureus was MRSA, with methicillin resistance also detected in Staphylococcus epidermidis, Staphylococcus haemolyticus and Mammaliicoccus sciuri. Through additional WGS analysis we report a cluster of M. sciuri in Pakistan identified between July-November 2017. Conclusions: In total we identified 14 different GPB bacterial species, however Staphylococci was dominant. These findings highlight the need of a prospective genomic epidemiology study to comprehensively assess the true burden of GPB neonatal sepsis focusing specifically on mechanisms of resistance and virulence across species and in relation to neonatal outcome

Neonatal sepsis and mortality in low-income and middle-income countries from a facility-based birth cohort: an international multisite prospective observational study

Background Neonatal sepsis is a primary cause of neonatal mortality and is an urgent global health concern, especially within low-income and middle-income countries (LMICs), where 99% of global neonatal mortality occurs. The aims of this study were to determine the incidence and associations with neonatal sepsis and all-cause mortality in facility-born neonates in LMICs. Methods The Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) study recruited mothers and their neonates into a prospective observational cohort study across 12 clinical sites from Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Data for sepsis-associated factors in the four domains of health care, maternal, birth and neonatal, and living environment were collected for all mothers and neonates enrolled. Primary outcomes were clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality in neonates during the first 60 days of life. Incidence proportion of livebirths for clinically suspected sepsis and laboratory-confirmed sepsis and incidence rate per 1000 neonate-days for all-cause mortality were calculated. Modified Poisson regression was used to investigate factors associated with neonatal sepsis and parametric survival models for factors associated with all-cause mortality. Findings Between Nov 12, 2015 and Feb 1, 2018, 29 483 mothers and 30 557 neonates were enrolled. The incidence of clinically suspected sepsis was 166·0 (95% CI 97·69–234·24) per 1000 livebirths, laboratory-confirmed sepsis was 46·9 (19·04–74·79) per 1000 livebirths, and all-cause mortality was 0·83 (0·37–2·00) per 1000 neonate-days. Maternal hypertension, previous maternal hospitalisation within 12 months, average or higher monthly household income, ward size (>11 beds), ward type (neonatal), living in a rural environment, preterm birth, perinatal asphyxia, and multiple births were associated with an increased risk of clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality. The majority (881 [72·5%] of 1215) of laboratory-confirmed sepsis cases occurred within the first 3 days of life. Interpretation Findings from this study highlight the substantial proportion of neonates who develop neonatal sepsis, and the high mortality rates among neonates with sepsis in LMICs. More efficient and effective identification of neonatal sepsis is needed to target interventions to reduce its incidence and subsequent mortality in LMICs. Funding Bill & Melinda Gates Foundation

Effects of antibiotic resistance, drug target attainment, bacterial pathogenicity and virulence, and antibiotic access and affordability on outcomes in neonatal sepsis: an international microbiology and drug evaluation prospective substudy (BARNARDS)

Background Sepsis is a major contributor to neonatal mortality, particularly in low-income and middle-income countries (LMICs). WHO advocates ampicillin–gentamicin as first-line therapy for the management of neonatal sepsis. In the BARNARDS observational cohort study of neonatal sepsis and antimicrobial resistance in LMICs, common sepsis pathogens were characterised via whole genome sequencing (WGS) and antimicrobial resistance profiles. In this substudy of BARNARDS, we aimed to assess the use and efficacy of empirical antibiotic therapies commonly used in LMICs for neonatal sepsis. Methods In BARNARDS, consenting mother–neonates aged 0–60 days dyads were enrolled on delivery or neonatal presentation with suspected sepsis at 12 BARNARDS clinical sites in Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Stillborn babies were excluded from the study. Blood samples were collected from neonates presenting with clinical signs of sepsis, and WGS and minimum inhibitory concentrations for antibiotic treatment were determined for bacterial isolates from culture-confirmed sepsis. Neonatal outcome data were collected following enrolment until 60 days of life. Antibiotic usage and neonatal outcome data were assessed. Survival analyses were adjusted to take into account potential clinical confounding variables related to the birth and pathogen. Additionally, resistance profiles, pharmacokinetic–pharmacodynamic probability of target attainment, and frequency of resistance (ie, resistance defined by in-vitro growth of isolates when challenged by antibiotics) were assessed. Questionnaires on health structures and antibiotic costs evaluated accessibility and affordability. Findings Between Nov 12, 2015, and Feb 1, 2018, 36 285 neonates were enrolled into the main BARNARDS study, of whom 9874 had clinically diagnosed sepsis and 5749 had available antibiotic data. The four most commonly prescribed antibiotic combinations given to 4451 neonates (77·42%) of 5749 were ampicillin–gentamicin, ceftazidime–amikacin, piperacillin–tazobactam–amikacin, and amoxicillin clavulanate–amikacin. This dataset assessed 476 prescriptions for 442 neonates treated with one of these antibiotic combinations with WGS data (all BARNARDS countries were represented in this subset except India). Multiple pathogens were isolated, totalling 457 isolates. Reported mortality was lower for neonates treated with ceftazidime–amikacin than for neonates treated with ampicillin–gentamicin (hazard ratio [adjusted for clinical variables considered potential confounders to outcomes] 0·32, 95% CI 0·14–0·72; p=0·0060). Of 390 Gram-negative isolates, 379 (97·2%) were resistant to ampicillin and 274 (70·3%) were resistant to gentamicin. Susceptibility of Gram-negative isolates to at least one antibiotic in a treatment combination was noted in 111 (28·5%) to ampicillin–gentamicin; 286 (73·3%) to amoxicillin clavulanate–amikacin; 301 (77·2%) to ceftazidime–amikacin; and 312 (80·0%) to piperacillin–tazobactam–amikacin. A probability of target attainment of 80% or more was noted in 26 neonates (33·7% [SD 0·59]) of 78 with ampicillin–gentamicin; 15 (68·0% [3·84]) of 27 with amoxicillin clavulanate–amikacin; 93 (92·7% [0·24]) of 109 with ceftazidime–amikacin; and 70 (85·3% [0·47]) of 76 with piperacillin–tazobactam–amikacin. However, antibiotic and country effects could not be distinguished. Frequency of resistance was recorded most frequently with fosfomycin (in 78 isolates [68·4%] of 114), followed by colistin (55 isolates [57·3%] of 96), and gentamicin (62 isolates [53·0%] of 117). Sites in six of the seven countries (excluding South Africa) stated that the cost of antibiotics would influence treatment of neonatal sepsis