Abstract. This study compares Escherichia coli pathotypes circulating among children in Pakistan during the floods of 2010 and 2011 and from sporadic cases outside flood affected areas. Using multiplex polymerase chain reaction 115 of 205 stool samples (56.29%) were positive for diarrheagenic E. coli from specimens taken during the floods compared with 50 of 400 (12.5%) stool samples being positive for sporadic cases. The E. coli pathotypes were categorized as Enteropathogenic E. coli 33 (28.69%) and 13 (26%), Enterotoxigenic E. coli 29 (25.21%) and 15 (30%), Enteroaggregative E. coli 21 (18.2%) and 18 (36%), Enterohemorrhagic E. coli 5 (4.34%) and 1 (2%) from flood and sporadic cases, respectively. Furthermore, patients co-infected with more than one pathotype were 26 (22.60%) and 3 (6%) from flood and sporadic cases, respectively. The study shows an unexpectedly high rate of isolation of E. coli pathotypes suggesting Pakistan as an endemic region that requires active surveillance particularly during flood periods.

CD4+T cells orchestrate adaptive immunity to circulating malaria parasites; yet cellular interactions and molecular mechanisms controlling Th1 and Tfh differentiation in the spleen remain be fully defined in vivo. Here, using a murine model of CD4-dependent immunity, we tested ifSlide-seqV2, a spatial transcriptomic method with near single-cell resolution, could determine the locations of multiple CD4+T cell subsets and potentially interacting cellular partners within the spleen during infection. Firstly,Slide-seqV2readily mapped splenic cellular structure and microanatomical change during infection. Next, computational integration with scRNA-seq reference datasets of splenocytes, stromal cells, and specifically of polyclonal CD4+T cells and B cells, mapped the relative locations of multiple cell-types within this dense tissue. scRNA-seq of B cells over time mapped emergence of germinal centre B cells, red-pulp located plasmablasts and atypical B cells, and uncovered a prolonged CD4+T-cell-independent, follicular bystander B cell response, marked by Sca-1 and Ly6C upregulation. scRNA-seq of activated, polyclonal CD4+T cells revealed their similarity to our previous TCR transgenic models. Importantly, spatial analysis revealed polyclonal Th1 cells co-localised with CXCL9/10-producing monocytes in the red pulp, while polyclonal Tfh-like cells were located close to CXCL13-expressing B cell follicles, consistent with our previous CXCR3/CXCR5 competition model of Th1/Tfh bifurcation. CRISPR/Cas9 disruption of either or both CXCR3 and CXCR5 in naivePlasmodium-specific CD4+T cells had unexpectedly minor effects on Th1 differentiation in vivo. Instead, CXCR5 was essential for maximising clonal expansion, suggesting a role for splenic CXCL13+cells in supporting CD4+T cell proliferation in malaria. Thus, spatial transcriptomics at near single-cell resolution was feasible in densely packed secondary lymphoid tissue, providing multiple insights into mechanisms controlling splenic polyclonal CD4+T cell and B cell differentiation during infection.

Children in malaria-endemic regions can experience multiplePlasmodiuminfections over a short period of time, within vitroCD4+T cell recall responses becoming more regulatory with increasing age and exposure. This suggests that repeated infection qualitatively changes CD4+T cells, although the heterogeneity and dynamics of these responses await systematic analysisin vivo. Here, we examined TCR transgenic PbTII and polyclonal CD4+T cells duringPlasmodiumre-infection in mice, in conjunction with scRNA-seq/TCR-seq and spatial transcriptomics at near single-cell resolution. PbTII cells gave rise to multiple antigen-experienced states in different areas of the spleen after primary infection and antimalarial treatment, including ongoing GC responses and T-cell zone memory. Upon re-infection, Th1-memory PbTII cells initiated a rapid effector response prior to proliferating, while GC Tfh cells of the same antigen specificity were entirely refractory within the same organ. Transcriptome dynamic modelling and network analysis of Th1 recall revealed a biphasic wave of RNA processing that firstly preceded immune effector transcription, and later accompanied cellular proliferation. Importantly, Th1 recall constituted a partial facsimile of primary Th1 responses, with no unique genes amongst the small subset of those upregulated upon re-infection. Finally, we noted a similar spectrum of antigen-experienced states and recall dynamics by polyclonal CD4+T cells with diverse TCRs. Therefore, during re-infection withPlasmodium, persisting GC Tfh cells remained unaltered transcriptionally, Tcm/Tfh-like cells exhibited minimal proliferation, and Th1-memory cells displayed a rapid, proliferating IL-10-producing Tr1 response consistent with a shift towards immune-regulation. These data highlight a broad spectrum of simultaneous CD4+T cell responses that occur in the spleen during re-infection with malaria parasites.HighlightsSplenic TCR transgenic CD4+T cells are highly heterogeneous prior to re-infection.Persisting GC Tfh cells are refractory to re-activation during re-infection.Th1-memory cells rapidly upregulate RNA processing prior to effector function and proliferation.Th1-recall is an imperfect but faithful facsimile of primary Th1 responses.A spectrum of recall states is observed in polyclonal CD4+T cells with diverse TCRs.

A caesarean scar pregnancy (CSP) is a rare form of ectopic pregnancy in which gestational sac is Implanted inside the previous caesarean scar. It is a life-threatening form of abnormal Implantation of embryo within the myometrium and fibrous tissue of the previous scar following caesarean section. Transvaginal sonography (TVS) is the gold standard for diagnosis of CSP. If CSP is diagnosed early and correctly by TVS then patient outcome is better. One third of the cases are misdiagnosed. A patient G2P1L1A0 with previous one caesarean section came with history of amenorrhea 3 months and, pain abdomen and spotting P/V since morning, she had an ultrasound report with her showing, 8 weeks of intra-uterine pregnancy with no cardiac activity. Impression was missed abortion. Patient was prepared for suction and evacuation under GA. During the procedure, patient started bleeding heavily, her vitals deteriorated, an emergency laparatomy was done and it was found to be a scar pregnancy. Scar was excised; the patient withstood the surgery well. Post-operative period was uneventful.