Outcome data from 15 years of cystic fibrosis newborn screening in a large UK region.

BACKGROUND: The West Midlands Newborn Bloodspot Screening Laboratory is one of 16 in the UK and serves two tertiary paediatric cystic fibrosis (CF) centres (Staffordshire Children's Hospital at Royal Stoke and Birmingham Children's Hospital). CF newborn bloodspot screening (NBS) in this region started in November 2006 prior to the UK national roll-out in 2007. It uses an immunoreactive trypsinogen (IRT)/DNA/IRT protocol. We report the outcomes from 15 years of CF screening. METHODS: The West Midlands CF NBS outcomes from 1 November 2006 to 31 October 2021 were reviewed. Clinical data were also obtained for babies referred to the CF centres as 'CF suspected'. RESULTS: 1 075 161 babies were screened, with 402 referred as 'CF suspected' and 205 identified as CF carriers. Of the 'CF suspected' babies, 268 were diagnosed with CF, 33 with CF screen positive, inconclusive diagnosis (CFSPID) and 17 as a CF carrier. Any CF-related diagnosis was excluded in 67. Outcome data were not available for 17, of whom 14 had died. Eighteen children with a negative CF NBS have subsequently been diagnosed with CF, 10 had meconium ileus and 8 were true 'affected not detected', presenting with respiratory symptoms or failure to thrive. This gives the West Midlands a CF birth prevalence of 1 in 4012 live births and the NBS protocol a sensitivity of 97.1% and a positive predictive value of 66.7%. CONCLUSIONS: This large regional data set has excellent case ascertainment and demonstrates successful performance of the CF NBS protocol, with low numbers identified as CFSPID or CF carriers.

Shigella sonnei O-Antigen Inhibits Internalization, Vacuole Escape, and Inflammasome Activation.

Two Shigella species, Shigella flexneri and Shigella sonnei, cause approximately 90% of bacterial dysentery worldwide. While S. flexneri is the dominant species in low-income countries, S. sonnei causes the majority of infections in middle- and high-income countries. S. flexneri is a prototypic cytosolic bacterium; once intracellular, it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. In contrast, little is known about the invasion, vacuole escape, and induction of pyroptosis during S. sonnei infection of macrophages. We demonstrate here that S. sonnei causes substantially less pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolic S. sonnei and hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-antigen (O-Ag), which in S. sonnei is contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the type 3 secretion system (T3SS) was required for vacuole escape. Our findings suggest that S. sonnei has adapted to an extracellular lifestyle by incorporating multiple layers of O-Ag onto its surface compared to other Shigella species.IMPORTANCE Diarrheal disease remains the second leading cause of death in children under five. Shigella remains a significant cause of diarrheal disease with two species, S. flexneri and S. sonnei, causing the majority of infections. S. flexneri are well known to cause cell death in macrophages, which contributes to the inflammatory nature of Shigella diarrhea. Here, we demonstrate that S. sonnei causes less cell death than S. flexneri due to a reduced number of bacteria present in the cell cytosol. We identify the O-Ag polysaccharide which, uniquely among Shigella spp., is present in two forms on the bacterial cell surface as the bacterial factor responsible. Our data indicate that S. sonnei differs from S. flexneri in key aspects of infection and that more attention should be given to characterization of S. sonnei infection.

Enteropathogenic Escherichia coli Stimulates Effector-Driven Rapid Caspase-4 Activation in Human Macrophages.

Microbial infections can stimulate the assembly of inflammasomes, which activate caspase-1. The gastrointestinal pathogen enteropathogenic Escherichia coli (EPEC) causes localized actin polymerization in host cells. Actin polymerization requires the binding of the bacterial adhesin intimin to Tir, which is delivered to host cells via a type 3 secretion system (T3SS). We show that EPEC induces T3SS-dependent rapid non-canonical NLRP3 inflammasome activation in human macrophages. Notably, caspase-4 activation by EPEC triggers pyroptosis and cytokine processing through the NLRP3-caspase-1 inflammasome. Mechanistically, caspase-4 activation requires the detection of LPS and EPEC-induced actin polymerization, either via Tir tyrosine phosphorylation and the phosphotyrosine-binding adaptor NCK or Tir and the NCK-mimicking effector TccP. An engineered E. coli K12 could reconstitute Tir-intimin signaling, which is necessary and sufficient for inflammasome activation, ruling out the involvement of other virulence factors. Our studies reveal a crosstalk between caspase-4 and caspase-1 that is cooperatively stimulated by LPS and effector-driven actin polymerization.