Whole-Genome Phylogenomic Heterogeneity of Neisseria gonorrhoeae Isolates with Decreased Cephalosporin Susceptibility Collected in Canada between 1989 and 2013

A large-scale, whole-genome comparison of Canadian Neisseria gonorrhoeae isolates with high-level cephalosporin MICs was used to demonstrate a genomic epidemiology approach to investigate strain relatedness and dynamics. Although current typing methods have been very successful in tracing short-chain transmission of gonorrheal disease, investigating the temporal evolutionary relationships and geographical dissemination of highly clonal lineages requires enhanced resolution only available through whole-genome sequencing (WGS). Phylogenomic cluster analysis grouped 169 Canadian strains into 12 distinct clades. While some N. gonorrhoeae multiantigen sequence types (NG-MAST) agreed with specific phylogenomic clades or subclades, other sequence types (ST) and closely related groups of ST were widely distributed among clades. Decreased susceptibility to extended-spectrum cephalosporins (ESC-DS) emerged among a group of diverse strains in Canada during the 1990s with a variety of nonmosaic penA alleles, followed in 2000/2001 with the penA mosaic X allele and then in 2007 with ST1407 strains with the penA mosaic XXXIV allele. Five genetically distinct ESC-DS lineages were associated with penA mosaic X, XXXV, and XXXIV alleles and nonmosaic XII and XIII alleles. ESC-DS with coresistance to azithromycin was observed in 5 strains with 23S rRNA C2599T or A2143G mutations. As the costs associated with WGS decline and analysis tools are streamlined, WGS can provide a more thorough understanding of strain dynamics, facilitate epidemiological studies to better resolve social networks, and improve surveillance to optimize treatment for gonorrheal infections.     

Effect of Variants of Penicillin-Binding Protein 2 on Cephalosporin and Carbapenem Susceptibilities in Neisseria gonorrhoeae

To characterize the relationship between penicillin-binding protein 2 (PBP2/penA) and susceptibility to extended-spectrum cephalosporins (ESCs) and carbapenem antibiotics, we compared 17 PBP2 variants in Neisseria gonorrhoeae. Nonmosaic and mosaic variants of PBP2 caused decreased susceptibility to ESCs and, to a lesser extent, to carbapenems. An A501P substitution in mosaic XXXIV_A501P conferred decreased susceptibility to ESCs but restored carbapenem susceptibility to wild-type levels. These results could aid the molecular surveillance of antimicrobial resistance to these agents.     

Treatment of vertebrobasilar fusiform aneurysms with Pipeline embolization device

Object

Treatment of complex intracranial aneurysms with Pipeline embolization device (PED) (ev3/Covidien Vascular Therapies) has gained recent popularity. One application of PEDs that is not well described in the literature is the utility and long-term safety in treatment of vertebrobasilar fusiform (VBF) aneurysms. Despite the advancements in endovascular therapy, VBF aneurysms continue to challenging pathology. The authors provide long-term follow-up of VBF aneurysms treated with PEDs.

Methods

We retrospectively reviewed four patients that were treated at Louisiana State University Health Sciences Center in Shreveport with PEDs for VBFs from 2012 to 2014. Each patient was discussed in a multidisciplinary setting between neurosurgeons and neurointerventionalists. Each patient underwent platelet function tests to ensure responsiveness to anti-platelet agents and was treated by one neurointerventionalist (HC). All patients were placed on aspirin and Plavix and were confirmed for therapeutic response prior to discharge.

Results

Follow-up ranged from 12 to 25 months, with a mean of 14.25 months. Two cases presented with a recurrence after the initial treatment, both of which required subsequent treatment. Of the four patients treated, one patient developed hemiparesis and three died.

Conclusion

Despite reports describing successful treatment of VBF aneurysms with PEDs, delayed complications after obliteration and remodeling can occur. We describe our institutional experience of VBFs treated with PEDs. Treatment of holobasilar fusiform aneurysms may carry a worse prognosis after treatment. Further long-term follow-up will provide a better understanding of this pathology.     

Stress distribution patterns of implant supported overdentures-analog versus finite element analysis: A comparative in-vitro study

Aims and Objectives:

The aim of this study was to asses & compare the load transfer characteristics of Ball/O-ring and Bar/Clip attachment systems in implant supported overdentures using analog and finite element analysis models.

Methodology:

For the analog part of the study, castable bar was used for the bar and clip attachment and a metallic housing with a rubber O-ring component was used for the ball/O-ring attachment. The stress on the implant surface was measured using the strain-gauge technique. For the finite element analysis, the model were fabricated and load applications were done in a similar manner as in analog study.

Results:

The difference between both the attachment systems was found to be statistically significant (P<0.001).

Conclusion:

Ball/O-ring attachment system transmitted lesser amount of stresses to the implants on the non-loading side, as compared to the Bar-Clip attachment system. When overall stress distribution is compared, the Bar-Clip attachment seems to perform better than the Ball/O-ring attachment, because the force was distributed better.Key Words: Ball/O-ring, Bar-Clip, finite element analysis, implant retained overdenture, overdenture attachments, strain gauges     

“Identification of Non-Tuberculous Mycobacterium by LPA (CM/AS) assay,HPLC and biochemical test: which is feasible for RNTCP?”

Introduction

Non-tuberculous mycobacteria (NTM) causing clinical disease have become increasingly common and more diverse. The development of fast, inexpensive, and reliable tests to identify nontuberculous mycobacteria is need of the hour especially under the Revised National TB Control Programme (RNTCP). The Aim of the study was to check the Diagnostic efficacy of the GenoType Mycobacterium CM/AS assay compared with HPLC and Biochemical Test for Identification of Non-Tuberculous Mycobacteria under the Revised Tuberculosis Control Programme.

Transcatheter aortic valve replacement outcomes in bicuspid compared to trileaflet aortic valves

Aim

TAVR in patients with bicuspid aortic valves (BAV) is more challenging compared to individuals with trileaflet aortic valves (TAV). BAV have been excluded from the large randomized clinical trials assessing transcatheter aortic valve replacements (TAVR) and has been considered as a relative contraindication to TAVR. To report the outcomes of TAVR in BAV and compare them to TAV in the National Inpatient Sample (NIS).

Mutational fitness landscapes reveal genetic and structural improvement pathways for a vaccine-elicited HIV-1 broadly neutralizing antibody

Vaccine-based elicitation of broadly neutralizing antibodies holds great promise for preventing HIV-1 transmission. However, the key biophysical markers of improved antibody recognition remain uncertain in the diverse landscape of potential antibody mutation pathways, and a more complete understanding of anti–HIV-1 fusion peptide (FP) antibody development will accelerate rational vaccine designs. Here we survey the mutational landscape of the vaccine-elicited anti-FP antibody, vFP16.02, to determine the genetic, structural, and functional features associated with antibody improvement or fitness. Using site-saturation mutagenesis and yeast display functional screening, we found that 1% of possible single mutations improved HIV-1 envelope trimer (Env) affinity, but generally comprised rare somatic hypermutations that may not arise frequently in vivo. We observed that many single mutations in the vFP16.02 Fab could enhance affinity >1,000-fold against soluble FP, although affinity improvements against the HIV-1 trimer were more measured and rare. The most potent variants enhanced affinity to both soluble FP and Env, had mutations concentrated in antibody framework regions, and achieved up to 37% neutralization breadth compared to 28% neutralization of the template antibody. Altered heavy- and light-chain interface angles and conformational dynamics, as well as reduced Fab thermal stability, were associated with improved HIV-1 neutralization breadth and potency. We also observed parallel sets of mutations that enhanced viral neutralization through similar structural mechanisms. These data provide a quantitative understanding of the mutational landscape for vaccine-elicited FP-directed broadly neutralizing antibody and demonstrate that numerous antigen-distal framework mutations can improve antibody function by enhancing affinity simultaneously toward HIV-1 Env and FP.

The tremendous circulating sequence diversity of HIV-1 and its capacity to evade host immunity pose unique challenges for vaccine design (reviewed in refs. 1, 2). Broadly neutralizing antibodies (bNAbs) identified from HIV-1 patients target conserved vulnerable epitopes on the HIV-1 envelope protein (Env) to prevent HIV-1 infection, and HIV-1 bNAb elicitation has become a major goal for HIV-1 vaccine design (3, 4). Several HIV-1 vulnerable epitopes have been described (5) including the following: the V1V2 apex (6, 7), the CD4-binding site (8–10), the membrane-proximal external region (11, 12), the glycan-V3 region (also known as N332 glycan supersite) (10, 13), the highly glycosylated region at the center of the silent face on the gp120 subunit (14), and the fusion peptide (FP), which is required for viral entry (15–17). Several complementary approaches seek to develop immunogens that elicit broadly neutralizing HIV-1 antibodies, with promising results (4, 18, 19). However, vaccine-elicited HIV-1 antibodies are often either less potent or less broad than many of the bNAbs identified from human patients. There is a pressing need to better understand bNAb developmental pathways and outline the genetic and structural antibody features that can provide enhanced neutralization breadth and potency for HIV-1 vaccines.Clinical data show that broadly neutralizing serum antibodies develop naturally in around 20% of individuals with chronic HIV-1 infection (20, 21) and that a smaller number of individuals show highly potent neutralization (22, 23). bNAbs develop via the accumulation of somatic hypermutations (SHM) and affinity maturation following their initial B-cell selection and expansion. While HIV-1–infected individuals may have high titers against HIV-1 antigens, the rarity of bNAbs suggests that the mutations acquired during bNAb development are correspondingly rare and/or that the mutational pathways to effective bNAb development are explored only in the context of chronic HIV-1 antigen exposure over years of viral infection (24). Evidence that high viral load is correlated with higher HIV-1 serum breadth and potency also suggests that larger numbers of sampled antibody maturation pathways are correlated with broad HIV-1 neutralization (20). A major question for HIV-1 vaccine design is thus how to quickly and effectively induce B-cell maturation to bNAbs from a small number of controlled immunizations.HIV-1 bNAbs are often highly somatically mutated (25–28), and reverting these mutations to antibody germline sequences results in drastic reductions of neutralization breadth and potency (9, 29–31). Complementarity determining regions (CDRs) are known to be mutational hotspots and are often in direct contact with antigen to enable recognition. Antibody framework region (FR) mutations can also modulate neutralization breadth and potency by altering the structural orientation, particularly at heavy:light interface alignments, and by altering the intrinsic flexibility of the paratope (16, 32–35). Not all observed bNAb somatic mutations are required for high neutralization breadth and potency (36), and a better understanding of the critical mutations and antibody structural features (both naturally elicited and vaccine-induced) to improve neutralization breadth and potency would enhance our understanding of bNAb development and guide efficient HIV-1 vaccine strategies (16, 17, 37–41).Among various immunization approaches reported for eliciting broadly neutralizing antibodies (16–19, 38–42), one strategy targets the HIV-1 FP epitope and has elicited 59 and 31% HIV-1 neutralization breadth in rhesus macaques and mice, respectively, against a broad panel of 208 HIV-1 strains (16, 17). Priming the immune response with soluble FP followed by three Env trimer boosts elicited antibodies with FP-targeted trimer recognition and broad HIV-1 neutralization; however, the structural mechanisms that enable effective anti-FP antibody maturation and pathway selection are not fully understood. Several key unanswered questions include the following: 1) What are the structural features of FP recognition vs. trimer recognition that may appropriately guide antibody development?; 2) What critical parameters control virus neutralization of FP-targeting bNAbs?; and 3) How do beneficial mutations fit into the entire landscape of possible mutational pathways, and what fraction of possible mutations provide increased affinity, neutralization breadth, and potency?To address these questions, we characterized the genetic and functional fitness landscape of an anti-FP bNAb and used biophysical and structural techniques to follow the mechanisms of antibody improvement. We implemented these screening strategies using the anti-FP bNAb vFP16.02, a vaccine-elicited antibody that neutralizes ∼30% of HIV-1 viral isolates (16). We applied yeast display and fluorescence-activated cell sorting (FACS) coupled with next-generation sequencing (NGS) to identify single mutations that enhanced binding or fitness to multiple HIV-1 BG505 Env trimer variants (Fig. 1). We mapped possible single mutations by their functional impacts and identified a panel of mutations with enhanced binding affinity and neutralization. Structural analyses of a subset of these mutations provided insights into the mechanisms of enhanced neutralization. These data confirm that several parallel mutational pathways exist for HIV-1 bNAb improvement and underscore the importance of improved Env trimer affinity for enhancing neutralization potency and breadth in HIV-1 vaccine designs.Open in a separate windowFig. 1.Experimental workflow for comprehensive functional analysis of all possible single mutations in an HIV-1 bNAb. SSM libraries were designed for VH and VL regions of the anti–HIV-1 FP bNAb vFP16.02. SSM libraries were cloned into yeast Fab surface display libraries and screened by FACS to determine the functional impact of each possible single mutation. Single-mutation display libraries were sorted for their affinity against a BG505 DS-SOSIP HIV-1 Env trimer with two different FP sequences. Sorted yeast libraries were prepped for NGS to enable quantitative variant tracking across three screening rounds; bioinformatic analyses of NGS data were used to interpret the functional impact of each possible amino acid mutation. Selected variants were characterized for neutralization activity and affinity against soluble FP and against HIV-1 Env. Structural analyses were performed to understand the mechanistic basis of anti–HIV-1 FP bNAb improvement.