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The Medicine Quality Research Group has published a new Medical Product Quality Report focussing on increasing issues around substandard and falsified (SF) COVID-19 vaccines. With the implementation of the key innovations of COVID-19 vaccines, there have been growing numbers of reports of SF vaccines in the public domain. Given the vital role they will play in ending the pandemic and protecting the global population but severe issues with equitable access, SF vaccines are highly likely to be a growing problem.
Stunting in the first year of life: Pathway analysis of a birth cohort
Malnutrition among infants aged below 6 months has been largely overlooked creating gaps in our understanding of factors underlying stunting in early infancy. Recent evidence suggests that pre-natal and early childhood factors may contribute more to driving childhood stunting than previously appreciated. The study was set up to examine pathways including parental and household characteristics, birth size and gestation, and illness in infancy with stunting at birth and months 3, 6 and 12 using an a priori hypothesized framework. It was a secondary analysis of a birth cohort of 1017 infants recruited from four health facilities in Burkina Faso and followed up for one year. Structural equation models (SEM) were generated to explore pathways to stunting at birth and months 3, 6 and 12. The prevalence of being stunted at birth and months 3, 6 and 12 was 7.4%, 23%, 20% and 18% respectively. The fractions of month 12 stunting attributable to being stunted at birth, months 3 and 6 were 11% (95%CI 5.0‒16%), 32% (95%CI 22‒41%) and 40% (95%CI 31‒49%) respectively. In the structural equation model, male sex and maternal characteristics had direct effects on stunting at birth and at 3 months, but not subsequently. Premature birth, twin birth and being stunted at a previous time point were directly associated with stunting at months 3, 6 and 12. Both maternal and paternal characteristics were directly associated with preterm birth. Non-exclusive breastfeeding had borderline positive direct effect on stunting at month 6 but not at month 12. The direct and indirect pathways identified in this study highlight the complex interlinks between child, maternal, paternal and household characteristics. Interventions tackling preterm birth, in utero growth, exclusive breastfeeding and maternal wellbeing may reduce stunting in the first year of life.
Frequent recombination of pneumococcal capsule highlights future risks of emergence of novel serotypes
AbstractCapsular diversity ofStreptococcus pneumoniaeconstitutes a major obstacle in eliminating the pneumococcal disease. Such diversity is genetically encoded by almost 100 variants of the capsule polysaccharide locus (cps). However, the evolutionary dynamics of the capsule – the target of the currently used vaccines – remains not fully understood. Here, using genetic data from 4,469 bacterial isolates, we foundcpsto be an evolutionary hotspot with elevated substitution and recombination rates. These rates were a consequence of altered selection at this locus, supporting the hypothesis that the capsule has an increased potential to generate novel diversity compared to the rest of the genome. Analysis of twelve serogroups revealed their complex evolutionary history, which was principally driven by recombination with other serogroups and other streptococci. We observed significant variation in recombination rates between different serogroups. This variation could only be partially explained by the lineage-specific recombination rate, the remaining factors being likely driven by serogroup-specific ecology and epidemiology. Finally, we discovered two previously unobserved mosaic serotypes in the densely sampled collection from Mae La, Thailand, here termed 10X and 21X. Our results thus emphasise the strong adaptive potential of the bacterium by its ability to generate novel serotypes by recombination.
Pneumococcal within-host diversity during colonization, transmission and treatment
AbstractCharacterizing the genetic diversity of pathogens within the host promises to greatly improve surveillance and reconstruction of transmission chains. For bacteria, it also informs our understanding of inter-strain competition and how this shapes the distribution of resistant and sensitive bacteria. Here we study the genetic diversity of Streptococcus pneumoniae within 468 infants and 145 of their mothers by deep sequencing whole pneumococcal populations from 3,761 longitudinal nasopharyngeal samples. We demonstrate that deep sequencing has unsurpassed sensitivity for detecting multiple colonization, doubling the rate at which highly invasive serotype 1 bacteria were detected in carriage compared with gold-standard methods. The greater resolution identified an elevated rate of transmission from mothers to their children in the first year of the child’s life. Comprehensive treatment data demonstrated that infants were at an elevated risk of both the acquisition and persistent colonization of a multidrug-resistant bacterium following antimicrobial treatment. Some alleles were enriched after antimicrobial treatment, suggesting that they aided persistence, but generally purifying selection dominated within-host evolution. Rates of co-colonization imply that in the absence of treatment, susceptible lineages outcompeted resistant lineages within the host. These results demonstrate the many benefits of deep sequencing for the genomic surveillance of bacterial pathogens.
Horizontal gene transfer rate is not the primary determinant of observed antibiotic resistance frequencies in Streptococcus pneumoniae
Resistance frequencies are best explained as the outcome of selection, irrespective of the rate at which resistance is acquired.
Antibiotic susceptibility of clinical Burkholderia pseudomallei isolates in northeast Thailand during 2015-2018 and the genomic characterization of β-lactam-resistant isolates.
Melioidosis is an often fatal infection in tropical regions caused by an environmental bacterium, Burkholderia pseudomallei Current recommended melioidosis treatment requires intravenous β-lactam antibiotics such as ceftazidime (CAZ), meropenem (MEM) or amoxicillin-clavulanic acid (AMC) and oral trimethoprim-sulfamethoxazole. Emerging antibiotic resistance could lead to therapy failure and high mortality. We performed a prospective multicentre study in northeast Thailand during 2015-2018 to evaluate antibiotic susceptibility and characterize β-lactam resistance in clinical B. pseudomallei isolates. Collection of 1,317 B. pseudomallei isolates from patients with primary and relapse infections were evaluated for susceptibility to CAZ, imipenem (IPM), MEM and AMC. β-lactam resistant isolates were confirmed by broth microdilution method and characterized by whole genome sequence analysis, penA expression and β-lactamase activity. The resistant phenotype was verified via penA mutagenesis. All primary isolates were IPM-susceptible but we observed two CAZ-resistant and one CAZ-intermediate resistant isolates, two MEM-less susceptible isolates, one AMC-resistant and two AMC-intermediate resistant isolates. One of 13 relapse isolates was resistant to both CAZ and AMC. Two isolates were MEM-less susceptible. Strains DR10212A (primary) and DR50054E (relapse) were multi-drug resistant. Genomic and mutagenesis analyses supplemented with gene expression and β-lactamase analyses demonstrated that CAZ-resistant phenotype was caused by PenA variants: P167S (N=2) and penA amplification (N=1). Despite the high mortality rate in melioidosis, our study revealed that B. pseudomallei isolates had a low frequency of β-lactam resistance caused by penA alterations. Clinical data suggest that resistant variants may emerge in patients during antibiotic therapy and be associated with poor response to treatment.
Genetic diversity, determinants, and dissemination ofBurkholderia pseudomalleilineages implicated in melioidosis in northeast Thailand
Melioidosis is an often-fatal neglected tropical disease caused by an environmental bacterium Burkholderia pseudomallei . However, our understanding of the disease-causing bacterial lineages, their dissemination, and adaptive mechanisms remains limited. To address this, we conducted a comprehensive genomic analysis of 1,391 B. pseudomallei isolates collected from nine hospitals in northeast Thailand between 2015 and 2018, and contemporaneous isolates from neighbouring countries, representing the most densely sampled collection to date. Our study identified three dominant lineages with unique gene sets enhancing bacterial fitness, indicating lineage-specific adaptation strategies. Crucially, recombination was found to drive lineage-specific gene flow. Transcriptome analyses of representative clinical isolates from each dominant lineage revealed heightened expression of lineage-specific genes in environmental versus infection conditions, notably under nutrient depletion, highlighting environmental persistence as a key factor in the success of dominant lineages. The study also revealed the role of environmental factors – slope of terrain, altitude, direction of rivers, and the northeast monsoons – in shaping B. pseudomallei geographical dispersal. Collectively, our findings highlight persistence in the environment as a pivotal element facilitating B. pseudomallei spread, and as a prelude to exposure and infection, thereby providing useful insights for informing melioidosis prevention and control strategies.
An Evolutionary Arms Race Between Burkholderia pseudomallei and Host Immune System: What Do We Know?
A better understanding of co-evolution between pathogens and hosts holds promise for better prevention and control strategies. This review will explore the interactions between Burkholderia pseudomallei, an environmental and opportunistic pathogen, and the human host immune system. B. pseudomallei causes "Melioidosis," a rapidly fatal tropical infectious disease predicted to affect 165,000 cases annually worldwide, of which 89,000 are fatal. Genetic heterogeneities were reported in both B. pseudomallei and human host population, some of which may, at least in part, contribute to inter-individual differences in disease susceptibility. Here, we review (i) a multi-host-pathogen characteristic of the interaction; (ii) selection pressures acting on B. pseudomallei and human genomes with the former being driven by bacterial adaptation across ranges of ecological niches while the latter are driven by human encounter of broad ranges of pathogens; (iii) the mechanisms that generate genetic diversity in bacterial and host population particularly in sequences encoding proteins functioning in host-pathogen interaction; (iv) reported genetic and structural variations of proteins or molecules observed in B. pseudomallei-human host interactions and their implications in infection outcomes. Together, these predict bacterial and host evolutionary trajectory which continues to generate genetic diversity in bacterium and operates host immune selection at the molecular level.
Developing insights into the mechanisms of evolution of bacterial pathogens from whole-genome sequences.
Evolution of bacterial pathogen populations has been detected in a variety of ways including phenotypic tests, such as metabolic activity, reaction to antisera and drug resistance and genotypic tests that measure variation in chromosome structure, repetitive loci and individual gene sequences. While informative, these methods only capture a small subset of the total variation and, therefore, have limited resolution. Advances in sequencing technologies have made it feasible to capture whole-genome sequence variation for each sample under study, providing the potential to detect all changes at all positions in the genome from single nucleotide changes to large-scale insertions and deletions. In this review, we focus on recent work that has applied this powerful new approach and summarize some of the advances that this has brought in our understanding of the details of how bacterial pathogens evolve.
Rapid whole-genome sequencing for investigation of a neonatal MRSA outbreak.
BackgroundIsolates of methicillin-resistant Staphylococcus aureus (MRSA) belonging to a single lineage are often indistinguishable by means of current typing techniques. Whole-genome sequencing may provide improved resolution to define transmission pathways and characterize outbreaks.MethodsWe investigated a putative MRSA outbreak in a neonatal intensive care unit. By using rapid high-throughput sequencing technology with a clinically relevant turnaround time, we retrospectively sequenced the DNA from seven isolates associated with the outbreak and another seven MRSA isolates associated with carriage of MRSA or bacteremia in the same hospital.ResultsWe constructed a phylogenetic tree by comparing single-nucleotide polymorphisms (SNPs) in the core genome to a reference genome (an epidemic MRSA clone, EMRSA-15 [sequence type 22]). This revealed a distinct cluster of outbreak isolates and clear separation between these and the nonoutbreak isolates. A previously missed transmission event was detected between two patients with bacteremia who were not part of the outbreak. We created an artificial "resistome" of antibiotic-resistance genes and demonstrated concordance between it and the results of phenotypic susceptibility testing; we also created a "toxome" consisting of toxin genes. One outbreak isolate had a hypermutator phenotype with a higher number of SNPs than the other outbreak isolates, highlighting the difficulty of imposing a simple threshold for the number of SNPs between isolates to decide whether they are part of a recent transmission chain.ConclusionsWhole-genome sequencing can provide clinically relevant data within a time frame that can influence patient care. The need for automated data interpretation and the provision of clinically meaningful reports represent hurdles to clinical implementation. (Funded by the U.K. Clinical Research Collaboration Translational Infection Research Initiative and others.).
Genetic characterisation of Malawian pneumococci prior to the roll-out of the PCV13 vaccine using a high-throughput whole genome sequencing approach.
BackgroundMalawi commenced the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) into the routine infant immunisation schedule in November 2011. Here we have tested the utility of high throughput whole genome sequencing to provide a high-resolution view of pre-vaccine pneumococcal epidemiology and population evolutionary trends to predict potential future change in population structure post introduction.MethodsOne hundred and twenty seven (127) archived pneumococcal isolates from randomly selected adults and children presenting to the Queen Elizabeth Central Hospital, Blantyre, Malawi underwent whole genome sequencing.ResultsThe pneumococcal population was dominated by serotype 1 (20.5% of invasive isolates) prior to vaccine introduction. PCV13 is likely to protect against 62.9% of all circulating invasive pneumococci (78.3% in under-5-year-olds). Several Pneumococcal Molecular Epidemiology Network (PMEN) clones are now in circulation in Malawi which were previously undetected but the pandemic multidrug resistant PMEN1 lineage was not identified. Genome analysis identified a number of novel sequence types and serotype switching.ConclusionsHigh throughput genome sequencing is now feasible and has the capacity to simultaneously elucidate serotype, sequence type and as well as detailed genetic information. It enables population level characterization, providing a detailed picture of population structure and genome evolution relevant to disease control. Post-vaccine introduction surveillance supported by genome sequencing is essential to providing a comprehensive picture of the impact of PCV13 on pneumococcal population structure and informing future public health interventions.
Sequence element enrichment analysis to determine the genetic basis of bacterial phenotypes.
Bacterial genomes vary extensively in terms of both gene content and gene sequence. This plasticity hampers the use of traditional SNP-based methods for identifying all genetic associations with phenotypic variation. Here we introduce a computationally scalable and widely applicable statistical method (SEER) for the identification of sequence elements that are significantly enriched in a phenotype of interest. SEER is applicable to tens of thousands of genomes by counting variable-length k-mers using a distributed string-mining algorithm. Robust options are provided for association analysis that also correct for the clonal population structure of bacteria. Using large collections of genomes of the major human pathogens Streptococcus pneumoniae and Streptococcus pyogenes, SEER identifies relevant previously characterized resistance determinants for several antibiotics and discovers potential novel factors related to the invasiveness of S. pyogenes. We thus demonstrate that our method can answer important biologically and medically relevant questions.
Phenotypic and genetic alterations of Burkholderia pseudomallei in patients during relapse and persistent infections.
The bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe tropical disease associated with high mortality and relapse and persistent infections. Treatment of melioidosis requires prolonged antibiotic therapy; however, little is known about relapse and persistent infections, particularly the phenotypic and genetic alterations of B. pseudomallei in patients. In this study, we performed pulsed-field gel electrophoresis (PFGE) to compare the bacterial genotype between the initial isolate and the subsequent isolate from each of 23 suspected recurrent and persistent melioidosis patients in Northeast Thailand. We used whole-genome sequencing (WGS) to investigate multilocus sequence types and genetic alterations of within-host strain pairs. We also investigated the bacterial phenotypes associated with relapse and persistent infections, including multinucleated giant cell (MNGC) formation efficiency and intracellular multiplication. We first identified 13 (1.2%) relapse, 7 (0.7%) persistent, and 3 (0.3%) reinfection patients from 1,046 survivors. Each of the 20 within-host strain pairs from patients with relapse and persistent infections shared the same genotype, suggesting that the subsequent isolates arise from the infecting isolate. Logistic regression analysis of clinical data revealed regimen and duration of oral antibiotic therapies as risk factors associated with relapse and persistent infections. WGS analysis demonstrated 17 within-host genetic alteration events in 6 of 20 paired isolates, including a relatively large deletion and 16 single-nucleotide polymorphism (stocktickerSNP) mutations distributed across 12 genes. In 1 of 20 paired isolates, we observed significantly increased cell-to-cell fusion and intracellular replication in the second isolate compared with the initial isolate from a patient with persistent infection. WGS analysis suggested that a non-synonymous mutation in the tssB-5 gene, which encoded an essential component of the type VI secretion system, may be associated with the increased intracellular replication and MNGC formation efficiency of the second isolate of the patient. This information provides insights into genetic and phenotypic alterations in B. pseudomallei in human melioidosis, which may represent a bacterial strategy for persistent and relapse infections.
A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance.
Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection-such as multiplexed detection for viral variant surveillance-may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool-CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured-may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.
Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a.
Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B. pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B. pseudomallei, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B. pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B. pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA.