No abstract

Neonatal sepsis is defined as a systemic infection within the first 28 days of life, with early-onset sepsis (EOS) occurring within the first 72h, although the definition of EOS varies in literature. Whilst the global incidence has dramatically reduced over the last decade, neonatal sepsis remains an important cause of neonatal mortality, highest in low- and middle-income countries (LMICs). Symptoms at the onset of neonatal sepsis can be subtle, and therefore EOS is often difficult to diagnose from clinical presentation and laboratory testing and blood cultures are not always conclusive or accessible, especially in resource limited countries. Although the World Health Organisation (WHO) currently advocates a ß-lactam, and gentamicin for first line treatment, availability and cost influence the empirical antibiotic therapy administered. Antibiotic treatment of neonatal sepsis in LMICs is highly variable, partially caused by factors such as cost of antibiotics (and who pays for them) and access to certain antibiotics. Antimicrobial resistance (AMR) has increased considerably over the past decade and this review discusses current microbiology data available in the context of the diagnosis, and treatment for EOS. Importantly, this review highlights a large variability in data availability, methodology, availability of diagnostics, and aetiology of sepsis pathogens.

Long-read sequencing (LRS) can resolve repetitive regions, a limitation of short read (SR) data. Reduced cost and instrument size has led to a steady increase in LRS across diagnostics and research. Here, we re-basecalled FAST5 data sequenced between 2018 and 2021 and analyzed the data in relation to gDNA across a large dataset (n = 200) spanning a wide GC content (25–67%). We examined whether re-basecalled data would improve the hybrid assembly, and, for a smaller cohort, compared long read (LR) assemblies in the context of antimicrobial resistance (AMR) genes and mobile genetic elements. We included a cost analysis when comparing SR and LR instruments. We compared the R9 and R10 chemistries and reported not only a larger yield but increased read quality with R9 flow cells. There were often discrepancies with ARG presence/absence and/or variant detection in LR assemblies. Flye-based assemblies were generally efficient at detecting the presence of ARG on both the chromosome and plasmids. Raven performed more quickly but inconsistently recovered small plasmids, notably a ∼15-kb Col-like plasmid harboring blaKPC. Canu assemblies were the most fragmented, with genome sizes larger than expected. LR assemblies failed to consistently determine multiple copies of the same ARG as identified by the Unicycler reference. Even with improvements to ONT chemistry and basecalling, long-read assemblies can lead to misinterpretation of data. If LR data are currently being relied upon, it is necessary to perform multiple assemblies, although this is resource (computing) intensive and not yet readily available/useable.

Legionella pneumophila, a Gram-negative bacillus, is the causative agent of Legionnaire’s disease, a form of severe community-acquired pneumonia. Infection can have high morbidity, with a high proportion of patients requiring ICU admission, and up to 10% mortality, which is exacerbated by the lack of efficacy of typical empirical antibiotic therapy against Legionella spp. The fastidious nature of the entire Legionellaceae family historically required inclusion of activated charcoal in the solid medium to remove growth inhibitors, which inherently interferes with accurate antimicrobial susceptibility determination, an acknowledged methodological shortfall, now rectified by a new solid medium that gives results comparable to those of microbroth dilution. Here, as an international Legionella community (with authors representing various international reference laboratories, countries and clinical stakeholders for diagnosis and treatment of legionellosis), we set out recommendations for the standardization of antimicrobial susceptibility testing methods, guidelines and reference strains to facilitate an improved era of antibiotic resistance determination.

Chorioamnionitis is a major risk factor for preterm birth and an independent risk factor for postnatal morbidity for which currently successful therapies are lacking. Emerging evidence indicates that the timing and duration of intra-amniotic infections are crucial determinants for the stage of developmental injury at birth. Insight into the dynamical changes of organ injury after the onset of chorioamnionitis revealed novel therapeutic windows of opportunity. Importantly, successful development and implementation of therapies in clinical care is currently impeded by a lack of diagnostic tools for early (prenatal) detection and surveillance of intra-amniotic infections. In the current study we questioned whether an intra-amniotic infection could be accurately diagnosed by a specific volatile organic compound (VOC) profile in exhaled breath of pregnant sheep. For this purpose pregnant Texel ewes were inoculated intra-amniotically with Ureaplasma parvum and serial collections of exhaled breath were performed for 6 days. Ureaplasma parvum infection induced a distinct VOC-signature in expired breath of pregnant sheep that was significantly different between day 0 and 1 vs. day 5 and 6. Based on a profile of only 15 discriminatory volatiles, animals could correctly be classified as either infected (day 5 and 6) or not (day 0 and 1) with a sensitivity of 83% and a specificity of 71% and an area under the curve of 0.93. Chemical identification of these distinct VOCs revealed the presence of a lipid peroxidation marker nonanal and various hydrocarbons including n-undecane and n-dodecane. These data indicate that intra-amniotic infections can be detected by VOC analyses of exhaled breath and might provide insight into temporal dynamics of intra-amniotic infection and its underlying pathways. In particular, several of these volatiles are associated with enhanced oxidative stress and undecane and dodecane have been reported as predictive biomarker of spontaneous preterm birth in humans. Applying VOC analysis for the early detection of intra-amniotic infections will lead to appropriate surveillance of these high-risk pregnancies, thereby facilitating appropriate clinical course of action including early treatment of preventative measures for pre-maturity-associated morbidities.

The adaptive immune system protects against infection via selection of specific antigen receptors on B-cells and T-cells. We studied the prevalent CD8 killer T-cell response mounted against SARS-CoV-2 Spike269-277 epitope YLQPRTFLL via the most frequent Human Leukocyte Antigen (HLA) class I worldwide, HLA A*02. The widespread Spike P272L mutation has arisen in five different SARS-CoV-2 lineages to date and was common in the B.1.177 lineage associated with establishing the second wave in Europe. The large CD8 T-cell response seen across a cohort of HLA A*02+ convalescent patients, comprising of over 120 different TCRs, failed to respond to the P272L variant suggesting that proline 272 dominates TCR contacts with this epitope. Additionally, sizable populations (0.01%-0.2%) of total CD8 T-cells from individuals vaccinated against SARS-CoV-2 stained with HLA A*02-YLQPRTFLL multimers but failed to bind to the P272L reagent. Viral escape at prevalent T-cell epitopes restricted by high frequency HLA may be particularly problematic when vaccine immunity is focussed on a single protein such as SARS-CoV-2 Spike and provides a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlights the urgent need for monitoring T-cell escape in new SARS-CoV-2 variants.

Objectives To evaluate the accuracy, susceptibility and specificity of MYCOPLASMA IST3, the next generation of the most popular culture-based in vitro diagnostic device designed to detect, identify and test the susceptibility of urogenital mycoplasma infections. Methods MYCOPLASMA IST3 was evaluated against culture- and molecular-based gold standard methodologies to detect, identify, enumerate and determine antimicrobial resistance for Mycoplasma hominis and Ureaplasma species in 516 clinical samples collected across France, Serbia and the UK. Sample types included vulvovaginal/endocervical or urethral swabs (dry swab or eSwab®), semen and urine samples, which included blinded analysis following addition of a panel of 80 characterized control strains. Results Overall species identification was excellent for both Ureaplasma spp. (98.4% sensitivity, 99.7% specificity) and M. hominis (95.7% sensitivity, 100% specificity) relative to combined colony morphology on agar and quantitative PCR standards. Non-dilution-based bacterial load estimation by the assay was accurate between 83.7% (M. hominis) and 86.3% (Ureaplasma spp.) of the time (increased to 94.2% and 100%, respectively, if ±10-fold variance was allowed) relative to colonies counted on agar. Resistance accuracy for Ureaplasma spp. varied from gold standards for only 11/605 of individual tests (major error rate = 1.8%) and for 14/917 individual tests for M. hominis (major error rate = 1.5%). Conclusions The redesigned MYCOPLASMA IST3 assay eliminated previous shortcomings by providing independent accurate resistance screening of M. hominis and Ureaplasma species, even in mixed infections, with CLSI-compliant thresholds. Specificity, sensitivity and enumeration estimates correlated closely with the confirmatory methods.

Background The burden of antibiotic resistant infection is mainly felt in low-to-middle income countries, where the rate of antimicrobial resistance is largely under-surveyed and under huge pressure from unregulated, disparate and often self-guided access to antimicrobials. Nosocomial infections from hospital environments have been shown to be a particularly prevalent source of multi-drug resistant strains, yet surveillance of hospital environmental contamination is often not investigated. Methods The study was prospective, observational and cross-sectional, sampling 231 high and low touch surfaces from 15th March to 13th April 2021, from five wards in the Cape Coast Teaching Hospital, Ghana. Microbial growth in the presence of vancomycin and either meropenem or cefotaxime was examined and bacterial species were identified by MALDI-TOF. The presence of common extended-spectrum β-lactamases (ESBL) and carbapenemase antimicrobial resistance genes (ARG) were identified through PCR screening, which were confirmed by phenotypic antimicrobial susceptibility determination. Isolates positive for carbapenem resistance genes were sequenced using a multi-platform approach. Results We recovered microbial growth from 99% of swabs (n = 229/231) plated on agar in the absence of antimicrobials. Multiple sites were found to be colonised with resistant bacteria throughout the hospital setting. Bacteria with multi-drug resistance and ARG of concern were isolated from high and low touch points with evidence of strain dissemination throughout the environment. A total of 21 differing species of bacteria carrying ARG were isolated. The high prevalence of Acinetobacter baumannii carrying blaNDM-1 observed was further characterised by whole genome sequencing and phylogenetic analysis to determine the relationship between resistant strains found in different wards. Conclusion Evidence of multiple clonal incursions of MDR bacteria of high sepsis risk were found in two separate wards for a regional hospital in Ghana. The prevalence of multiple blaNDM carrying species in combination with combinations of ESBLs was particularly concerning and unexpected in Africa. We also identify strains carrying tet(X3), blaVIM-5 or blaDIM-1 showing a high diversity of carbapenamases present as a reservoir in a hospital setting. Findings of multi-drug resistant bacteria from multiple environmental sites throughout the hospital will inform future IPC practices and aid research prioritisation for AMR in Ghana.