Paediatric antimicrobial stewardship programmes in the UK's regional children's hospitals.

A survey was conducted in UK regional children's hospitals with paediatric intensive care and paediatric infectious disease (PID) departments to describe the characteristics of paediatric antimicrobial stewardship (PAS) programmes. A structured questionnaire was sent to PAS coordinators. 'Audit and feedback' was implemented in 13 out of 17 centres. Microbiology-led services were more likely to implement antimicrobial restriction (75% vs 33% in PID-led services), to focus on broad-spectrum antibiotics, and to review patients with positive blood cultures. PID-led services were more likely to identify patients from e-prescribing or drug charts and review all antimicrobials. A PAS network has been established.

Bacteremia in Childhood Life-Threatening Infections in Urban Gambia: EUCLIDS in West Africa.

BACKGROUND: The limited availability of microbiology services in sub-Saharan Africa impedes accurate diagnosis of bacterial pathogens and understanding of trends in prevalence and antibiotic sensitivities. We aimed to characterize bacteremia among hospitalized children in The Gambia and to identify factors associated with bacteremia and mortality. METHODS: We prospectively studied children presenting with suspected severe infection to 2 urban hospitals in The Gambia, between January 2013 and September 2015. Demographic and anthropometric data, clinical features, management, and blood culture results were documented. Urine screens for antibiotic activity were performed in a subset of participants. RESULTS: Of 411 children enrolled (median age, 29 months; interquartile range, 11-82), 79.5% (325 of 409) reported prehospital antibiotic use. Antimicrobial activity by urinary screen for antibiotic activity was detected in 70.8% (n = 80 of 113). Sixty-six bacterial pathogens were identified in 65 (15.8%) participants and Staphylococcus aureus predominated. Gram-positive organisms were more commonly identified than Gram-negative (P < .01). Antibiotic resistance against first-line antimicrobials (ampicillin and gentamicin) was common among Gram-negative bacteria (39%; range, 25%-100%). Factors significantly associated with bacteremia included the following: gender, hydration status, musculoskeletal examination findings, admission to the Medical Research Council The Gambia at London School of Hygiene & Tropical Medicine hospital, and meeting sepsis criteria. Those associated with increased mortality were presence of a comorbidity, clinical pallor, tachypnea, and altered consciousness. Tachycardia was associated with reduced mortality. CONCLUSIONS: The bacteremia rate in children with suspected childhood life-threatening infectious diseases in The Gambia is high. The pattern of pathogen prevalence and antimicrobial resistance has changed over time compared with previous studies illustrating the importance of robust bacterial surveillance programs in resource-limited settings.

Rat tapasin: cDNA cloning and identification as a component of the class I MHC assembly complex.

During the assembly of major histocompatibility complex (MHC) class I molecules transient associations are formed with the endoplasmic reticulum resident chaperones calnexin and calreticulin, ERp57 oxidoreductase, and also with tapasin, the latter mediating binding of the class I molecules to the transporter associated with antigen processing (TAP). We report here the isolation of a cDNA encoding rat tapasin from a DA (RT1av1) library. The cDNA encodes a proline-rich (11.3%) polypeptide of 464 residues with a potential ER-retention KK motif at its COOH-terminus, and a predicted molecular mass of 48 kDa. Matrix-assisted laser-desorption ionisation (MALDI) mass spectrometry of peptides derived from in-gel tryptic digestion of a TAP-associated protein match regions of the predicted translation product. A species of the correct molecular mass and predicted pl was also identified in association with radiolabelled immunoprecipitates of the rat TAP complex analysed by two-dimensional gel electrophoresis. This confirms rat tapasin as a component of the rat MHC class I assembly complex.

Physical map of human 6p21.2-6p21.3: region flanking the centromeric end of the major histocompatibility complex.

We have physically mapped and cloned a 2.5-Mb chromosomal segment flanking the centromeric end of the major histocompatibility complex (MHC). We characterized in detail 27 YACs, 144 cosmids, 51 PACs, and 5 BACs, which will facilitate the complete genomic sequencing of this region of chromosome 6. The contig contains the genes encoding CSBP, p21, HSU09564 serine kinase, ZNF76, TCP-11, RPS10, HMGI(Y), BAK, and the human homolog of Tctex-7 (HSET). The GLO1 gene was mapped further centromeric in the 6p21.2-6p21.1 region toward TCTE-1. The gene order of the GLO1-HMGI(Y) segment in respect to the centromere is similar to the gene order in the mouse t-chromosome distal inversion, indicating that there is conservation in gene content but not gene order between humans and mice in this region. The close linkage of the BAK and CSBP genes to the MHC is of interest because of their possible involvement in autoimmune disease.

TAPASIN, DAXX, RGL2, HKE2 and four new genes (BING 1, 3 to 5) form a dense cluster at the centromeric end of the MHC.

TAPASIN, a gene recently shown to be required for antigen presentation through MHC class I molecules, is located 180 kbp centromeric of HLA-DP in a region linked to several diseases, and associated with altered developmental phenotypes in the mouse. We present the genomic analysis of a 70 kbp gene-dense segment flanking the TAPASIN locus, including sequence, structure and preliminary characterisation of seven additional genes. BING1 is a Zn finger gene containing a POZ motif. BING3 is similar to myosin regulatory light chain. BING4 shows homologies only to hypothetical yeast and Caenorhabditis elegans proteins. BING5 is found within an intron of BING4 on the complementary strand, and encodes a molecule with no homologies to database proteins. Another three genes were identified whose full sequence was not previously known; namely, RGL2, DAXX (BING2) and HKE2. RGL2 encodes an effector of Ras, homologous to the mouse RalGDS protein, Rlf. DAXX encodes an effector of Fas that stimulates apoptosis through the Jun kinase (JNK) pathway. The location of DAXX is of interest given the linkage of autoimmune disease to the MHC and to apoptosis.

Genomic structure and domain organisation of the human Bak gene.

The Bcl-2 homologue, Bak, is a potent inducer of apoptosis. FISH data presented here located the gene to 6p21.3. Mapping was consistent with its location centromeric of the HSET locus and approximately 400kb from the MHC. The construction of a contig of genomic clones across the locus facilitated the sequencing of a PAC containing the gene. Comparison of the gene structure to functional and physical domains revealed a good agreement between the physical structure and the intron-exon organisation. The position of a single intron was conserved in comparison to other members of the Bcl-2 family, namely Bax, CED-9, Bcl-X and Bcl-2, but all other introns were displaced, consistent with a divergent phylogeny.

Genomic analysis of the Tapasin gene, located close to the TAP loci in the MHC.

The Tapasin molecule is a member of the immunoglobulin (Ig) superfamily required for the association of TAP transporters and MHC class I heterodimers in the endoplasmic reticulum. In this study, the Tapasin gene was precisely mapped in relation to the MHC. The gene was centromeric of the HLA-DP locus between the HSET and HKE1.5 genes and within 500 kbp of the TAP1 and TAP2 genes. A homologous mouse EST was mapped to a syntenic position on chromosome 17, centromeric of the H-2 K locus. Similarly, the rat Tapasin gene was shown to be in an equivalent location with respect to the RT1.A locus. The localization of Tapasin, TAP, LMP and class I genes within such a short distance of each other on the chromosome implies some regulatory or functional significance. We determined the Tapasin gene sequence for comparison of its structure to that of other Ig superfamily members, such as MHC class I genes. The IgC domain was encoded by a separate exon. However, the positions of the other introns were not characteristic of other Ig superfamily genes, indicating that Tapasin has a distinct phylogeny.

A critical role for tapasin in the assembly and function of multimeric MHC class I-TAP complexes.

Newly assembled major histocompatibility complex (MHC) class I molecules, together with the endoplasmic reticulum chaperone calreticulin, interact with the transporter associated with antigen processing (TAP) through a molecule called tapasin. The molecular cloning of tapasin revealed it to be a transmembrane glycoprotein encoded by an MHC-linked gene. It is a member of the immunoglobulin superfamily with a probable cytoplasmic endoplasmic reticulum retention signal. Up to four MHC class I-tapasin complexes were found to bind to each TAP molecule. Expression of tapasin in a negative mutant human cell line (220) restored class I-TAP association and normal class I cell surface expression. Tapasin expression also corrected the defective recognition of virus-infected 220 cells by class I-restricted cytotoxic T cells, establishing a critical functional role for tapasin in MHC class I-restricted antigen processing.