High-throughput multiplex HLA genotyping by next-generation sequencing using multi-locus individual tagging.

BACKGROUND: Unambiguous human leukocyte antigen (HLA) typing is important in transplant matching and disease association studies. High-resolution HLA typing that is not restricted to the peptide-binding region can decrease HLA allele ambiguities. Cost and technology constraints have hampered high-throughput and efficient high resolution unambiguous HLA typing. We have developed a method for HLA genotyping that preserves the very high-resolution that can be obtained by next-generation sequencing (NGS) but also achieves substantially increased efficiency. Unambiguous HLA-A, B, C and DRB1 genotypes can be determined for 96 individuals in a single run of the Illumina MiSeq. RESULTS: Long-range amplification of full-length HLA genes from four loci was performed in separate polymerase chain reactions (PCR) using primers and PCR conditions that were optimized to reduce co-amplification of other HLA loci. Amplicons from the four HLA loci of each individual were then pooled and subjected to enzymatic library generation. All four loci of an individual were then tagged with one unique index combination. This multi-locus individual tagging (MIT) method combined with NGS enabled the four loci of 96 individuals to be analyzed in a single 500 cycle sequencing paired-end run of the Illumina-MiSeq. The MIT-NGS method generated sequence reads from the four loci were then discriminated using commercially available NGS HLA typing software. Comparison of the MIT-NGS with Sanger sequence-based HLA typing methods showed that all the ambiguities and discordances between the two methods were due to the accuracy of the MIT-NGS method. CONCLUSIONS: The MIT-NGS method enabled accurate, robust and cost effective simultaneous analyses of four HLA loci per sample and produced 6 or 8-digit high-resolution unambiguous phased HLA typing data from 96 individuals in a single NGS run.

HLA class I, KIR, and genome-wide SNP diversity in the RV144 Thai phase 3 HIV vaccine clinical trial.

RV144 is the first phase 3 HIV vaccine clinical trial to demonstrate efficacy. This study consisted of more than 8,000 individuals in each arm of the trial, representing the four major regions of Thailand. Human leukocyte antigen (HLA) class I and killer cell immunoglobulin-like receptor (KIR) genes, as well as 96 genome-wide ancestry informative markers (AIMs) were genotyped in 450 placebo HIV-1-uninfected individuals to identify the immunogenetic diversity and population structure of this cohort. High-resolution genotyping identified the common HLA alleles as A*02:03, A*02:07, A*11:01, A*24:02, A*24:07, A*33:03, B*13:01, B*15:02, B*18:01, B*40:01, B*44:03, B*46:01, B*58:01, C*01:02, C*03:02, C*03:04, C*07:01, C*07:02, C*07:04, and C*08:01. The most frequent three-loci haplotype was B*46:01-C*01:02-A*02:07. Framework genes KIR2DL4, 3DL2, and 3DL3 were present in all samples, and KIR2DL1, 2DL3, 3DL1, 2DS4, and 2DP1 occurred at frequencies greater than 90 %. The combined HLA and KIR profile suggests admixture with neighboring Asian populations. Principal component and correspondence analyses comparing the RV144 samples to the phase 3 International HapMap Project (HapMap3) populations using AIMs corroborated these findings. Structure analyses identified a distinct profile in the Thai population that did not match the Asian or other HapMap3 samples. This shows genetic variability unique to Thais in RV144, making it essential to take into account population stratification while performing genetic association studies. The overall analyses from all three genetic markers indicate that the RV144 samples are representative of the Thai population. This will inform subsequent host genetic analyses in the RV144 cohort and provide insight for future genetic association studies in the Thai population.

A single VHH-based toxin-neutralizing agent and an effector antibody protect mice against challenge with Shiga toxins 1 and 2.

Shiga toxin-producing Escherichia coli (STEC) is a major cause of severe food-borne disease worldwide, and two Shiga toxins, Stx1 and Stx2, are primarily responsible for the serious disease consequence, hemolytic-uremic syndrome (HUS). Here we report identification of a panel of heavy-chain-only antibody (Ab) V(H) (VHH) domains that neutralize Stx1 and/or Stx2 in cell-based assays. VHH heterodimer toxin-neutralizing agents containing two linked Stx1-neutralizing VHHs or two Stx2-neutralizing VHHs were generally much more potent at Stx neutralization than a pool of the two-component monomers tested in cell-based assays and in vivo mouse models. We recently reported that clearance of toxins can be promoted by coadministering a VHH-based toxin-neutralizing agent with an antitag monoclonal antibody (MAb), called the "effector Ab," that indirectly decorates each toxin molecule with four Ab molecules. Decoration occurs because the Ab binds to a common epitopic tag present at two sites on each of the two VHH heterodimer molecules that bind to each toxin molecule. Here we show that coadministration of effector Ab substantially improved the efficacy of Stx toxin-neutralizing agents to prevent death or kidney damage in mice following challenge with Stx1 or Stx2. A single toxin-neutralizing agent consisting of a double-tagged VHH heterotrimer--one Stx1-specific VHH, one Stx2-specific VHH, and one Stx1/Stx2 cross-specific VHH--was effective in preventing all symptoms of intoxication from Stx1 and Stx2 when coadministered with effector Ab. Overall, the availability of simple, defined, recombinant proteins that provide cost-effective protection against HUS opens up new therapeutic approaches to managing disease.

Selling tobacco: A comprehensive analysis of the U.S. tobacco advertising landscape.

OBJECTIVES: To describe advertising tactics of cigarette, e-cigarette, little cigar/cigarillo and smokeless tobacco manufacturers. METHODS: We conducted a content analysis of tobacco 827 ads run in the US in 2016. Ads were double coded by trained coders across ten domains: promotions, web/social media presence, use cues, warnings and disclaimers, descriptors, claims, activities, setting, imagery, and themes. RESULTS: Cigarette ads relied on promotional tactics like discounts and sweepstakes and featured links to websites and mobile apps, all of which can increase brand loyalty and customer engagement. E-cigarette ads used tactics that appear to target new consumers, such as highlighting the product's qualities and modeling product use. Little cigar/cigarillo ads often positioned the product as social and featured music, urban and nightlife settings. Smokeless tobacco ads frequently featured themes, activities and settings stereotypically thought of as masculine. CONCLUSIONS: The tactics used to advertise tobacco products can help generate new consumers, encourage product/brand switching, and escalate use among current users. Understanding how different products are advertised can inform the Food and Drug Administration's regulatory efforts, and tobacco counter-marketing campaigns.

A primer in the evaluation of quality in acute care settings.

Nurses are in a unique position to evaluate acute care quality. The authors describe common problems and challenges related to selection of variables and choice of databases for quality evaluations. Practical strategies to address these concerns also are discussed.

Friday night in the pediatric emergency department: a simulated exercise to promote clinical reasoning in the classroom.

High acuity in client populations has placed increasing demands on novice nurses to apply concepts of clinical reasoning to nursing practice. To assist nursing students in developing clinical reasoning skills, an exercise called "Friday Night in the Pediatric Emergency Department" was designed to give students real-time experiences in triage situations using a simulated clinical experience in a classroom setting. The author describes the exercise and its impact on the clinical reasoning skills of undergraduate nursing students.

Advanced practice role characteristics of the community/public health nurse specialist.

The purpose of this qualitative study was to describe the advanced practice role of nurses with master's degrees in community/public health nursing using their experiences and perspectives. The purposive sample consisted of 10 nurses who had master's degrees in community/public health nursing and were working in a variety of community health settings. Data were collected using audiotaped interviews and 1-day observations of study participants in their workplaces. An editing analysis technique was used to analyze the data. Findings indicated that role characteristics included advocacy and policy setting at the organizational, community, and state levels; a leadership style centered on empowerment; a broad sphere of influence; and high-level skills in large-scale program planning, project management, and building partnerships. Results provide important descriptive data about significant aspects of the advanced practice role of nurses with master's degrees in community/public health nursing.

Sheep monoclonal antibodies prevent systemic effects of botulinum neurotoxin A1.

Botulinum neurotoxin (BoNT) is responsible for causing botulism, a potentially fatal disease characterized by paralysis of skeletal muscle. Existing specific treatments include polyclonal antisera derived from immunized humans or horses. Both preparations have similar drawbacks, including limited supply, risk of adverse effects and batch to batch variation. Here, we describe a panel of six highly protective sheep monoclonal antibodies (SMAbs) derived from sheep immunized with BoNT/A1 toxoid (SMAbs 2G11, 4F7) or BoNT/A1 heavy chain C-terminus (HcC) (SMAbs 1G4, 5E2, 5F7, 16F9) with or without subsequent challenge immunization with BoNT/A1 toxin. Although each SMAb bound BoNT/A1 toxin, differences in specificity for native and recombinant constituents of BoNT/A1 were observed. Structural differences were suggested by pI (5E2 = 8.2; 2G11 = 7.1; 4F7 = 8.8; 1G4 = 7.4; 5F7 = 8.0; 16F9 = 5.1). SMAb protective efficacy vs. 10,000 LD50 BoNT/A1 was evaluated using the mouse lethality assay. Although not protective alone, divalent and trivalent combinations of SMabs, IG4, 5F7 and/or 16F9 were highly protective. Divalent combinations containing 0.5­4 µg/SMAb (1­8 µg total SMAb) were 100% protective against death with only mild signs of botulism observed; relative efficacy of each combination was 1G4 + 5F7 > 1G4 + 16F9 >> 5F7 + 16F9. The trivalent combination of 1G4 + 5F7 + 16F9 at 0.25 µg/SMAb (0.75 µg total SMAb) was 100% protective against clinical signs and death. These results reflect levels of protective potency not reported previously.

A novel strategy for development of recombinant antitoxin therapeutics tested in a mouse botulism model.

Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant 'targeting agent' that binds a toxin at two unique sites and a 'clearing Ab' that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab V(H) (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit.

Voices Along the Journey: Midwives' Perceptions of Implementing the CenteringPregnancy Model of Prenatal Care.

The purpose of this study was to describe the reported perceptions of six midwife participants at different stages of their engagement in a multiphase process of adopting a new model of prenatal care. Midwives were interviewed at five different stages during the process of implementing CenteringPregnancy, a model of group prenatal care. The research methodology used in this study was phenomenology. The conceptual framework for exploring the participants' perceptions was based on the Institute for Healthcare Improvement's patient-centered model and on the International Institute for Restorative Practices' empowerment model. The five themes that emerged from the midwives' experiences mirrored the stages of change health education model. Suggestions for the implementation and sustainability of the CenteringPregnancy model of care are provided based on the five themes that emerged from this study's findings.

Evaluating healthcare quality using natural language processing.

Consistent monitoring for quality indicators as adverse events or missed screening opportunities remains a difficult proposition for most healthcare organizations. Much of the clinical data needed for quality reports is imbedded in narrative reports in the electronic health record. Narrative data most often require costly retrieval by manual data extraction. NUD*IST, a qualitative research computer program, was used as an automated natural Language processing tool to extract and code data for analysis of screening and treatment for breast cancer. The study method demonstrated acceptable Levels of precision and recall compared to large-scale natural Language processing programs.

Comparison of selected outcomes of CenteringPregnancy versus traditional prenatal care.

The purpose of this study was to assess the effect of traditional prenatal care versus a group model of care, CenteringPregnancy, on maternal knowledge of pregnancy, social support, health locus of control, and satisfaction. The CenteringPregnancy model of care gives patients extended time with the provider in a group setting. The study used a two-group, pretest/posttest design of pregnant women who elected either the traditional approach to prenatal care (n = 48) or the Centering approach (n = 50). The results of this study showed statistically significant differences between the groups on posttest in knowledge of pregnancy. For social support and health locus of control, the high scores at pretest contributed to a ceiling effect, which limited the potential for change. Additional research is needed to assess health status outcomes among women electing alternative approaches to prenatal care.

Evaluating quality of primary care using the electronic medical record.

Healthcare organizations are under increasing pressure to evaluate and report the level of quality in their health services. The electronic medical record (EMR) has been used in acute care settings to provide clinical data for quality evaluations. The implementation of the EMR in primary care settings is a more recent development, and as a result, the EMR has not been widely used to evaluate quality in primary care. Little research exists that uses the primary care medical record as a source of data. What remains to be seen is the extent to which EMRs contain the variables needed to address quality of primary care. This article describes a study that investigated the viability of the EMR as a database for evaluating quality in a women's primary health clinic.

Emergency preparedness and bioterrorism response: development of an educational program for public health personnel.

Public health departments are under increasing pressure to provide emergency preparedness and bioterrorism response education to public health personnel. The challenge that health departments face is to provide cost-efficient, timely education to a large number of multidisciplinary personnel. This article describes an innovative strategy for providing this education to public health personnel using the health department's intranet system. The intranet system provided confidential information specific to the staff role and allowed for concurrent access to the program by multiple individuals at different service sites. Knowledge acquisition was tested through short multiple-choice questions that followed the specific information modules. The intranet system faced a number of challenges during the pilot-testing phase, primarily related to changes in the role of the public health nurse and limitations in funding and public health staff to maintain and monitor the bioterrorism response program and the intranet system. The design of the program may prove useful for other public health organizations when a need exists for quick delivery of information to a large number of personnel. It may especially be useful in providing basic emergency preparedness and bioterrorism education to new personnel in health departments.