Phasing of de novo mutations using a scaled-up multiple amplicon long-read sequencing approach.

De novo mutations (DNMs) play an important role in severe genetic disorders that reduce fitness. To better understand their role in disease, it is important to determine the parent-of-origin and timing of mutational events that give rise to these mutations, especially in sex-specific developmental disorders such as male infertility. However, currently available short-read sequencing approaches are not ideally suited for phasing, as this requires long continuous DNA strands that span both the DNM and one or more informative single-nucleotide polymorphisms. To overcome these challenges, we optimized and implemented a multiplexed long-read sequencing approach using Oxford Nanopore technologies MinION platform. We focused on improving target amplification, integrating long-read sequenced data with high-quality short-read sequence data, and developing an anchored phasing computational method. This approach handled the inherent phasing challenges of long-range target amplification and the normal accumulation of sequencing error associated with long-read sequencing. In total, 77 of 109 DNMs (71%) were successfully phased and parent-of-origin identified. The majority of phased DNMs were prezygotic (90%), the accuracy of which is highlighted by an average mutant allele frequency of 49.6% and standard error of 0.84%. This study demonstrates the benefits of employing an integrated short-read and long-read sequencing approach for large-scale DNM phasing.

Focused assessment with sonography for trauma in predicting early surgical intervention in hemodynamically unstable children with blunt abdominal trauma.

OBJECTIVES: The predictive accuracy and clinical role of the focused assessment with sonography for trauma (FAST) exam in pediatric blunt abdominal trauma are uncertain. This study investigates the performance of the emergency department (ED) FAST exam to predict early surgical intervention and subsequent free fluid (FF) in pediatric trauma patients. METHODS: Pediatric level 1 trauma patients ages 0 to 15 years with blunt torso trauma at a single trauma center were retrospectively reviewed. After stratification by initial hemodynamic (HD) instability, the association of a positive FAST with (1) early surgical intervention, defined as operative management (laparotomy or open pericardial window) or angiography within 4 hours of ED arrival and (2) presence of FF during early surgical intervention was determined. RESULTS: Among 508 salvageable pediatric trauma patients with an interpreted FAST exam, 35 (6.9%) had HD instability and 98 (19.3%) were FAST positive. A total of 42 of 508 (8.3%) patients required early surgical intervention, and the sensitivity and specificity of FAST predicting early surgical intervention were 59.5% and 84.3%, respectively. The specificity and positive predictive value of FF during early surgical intervention in FAST-positive HD unstable patients increased from 50% and 90.9% at 4 hours after ED arrival to 100% and 100% at 2 hours after ED arrival, respectively. CONCLUSIONS: In this large series of injured children, a positive FAST exam improves the ability to predict the need for early surgical intervention, and accuracy is greater for FF in HD unstable patients 2 hours after arrival to the ED.

Knowledge acquisition and retention following Saving Children's Lives course for healthcare providers in Botswana: a longitudinal cohort study.

OBJECTIVES: Millions of children die every year from serious childhood illnesses. Most deaths are avertable with access to quality care. Saving Children's Lives (SCL) includes an abbreviated high-intensity training (SCL-aHIT) for providers who treat serious childhood illnesses. The objective of this study was to examine the impact of SCL-aHIT on knowledge acquisition and retention of providers. SETTING: 76 participating centres who provide primary and secondary care in Kweneng District, Botswana. PARTICIPANTS: Doctors and nurses expected by the District Health Management Team to provide initial care to seriously ill children, completed SCL-aHIT between January 2014 and December 2016, submitted demographic data, course characteristics and at least one knowledge assessment. METHODS: Retrospective, cohort study. Planned and actual primary outcome was adjusted acquisition (change in total knowledge score immediately after training) and retention (change in score at 1, 3 and 6 months), secondary outcomes were pneumonia and dehydration subscores. Descriptive statistics and linear mixed models with random intercept and slope were conducted. Relevant institutional review boards approved this study. RESULTS: 211 providers had data for analysis. Cohort was 91% nurses, 61% clinic/health postbased and 45% pretrained in Integrated Management of Childhood Illness (IMCI). A strong effect of SCL-aHIT was seen with knowledge acquisition (+24.56±1.94, p<0.0001), and loss of retention was observed (-1.60±0.67/month, p=0.018). IMCI training demonstrated no significant effect on acquisition (+3.58±2.84, p=0.211 or retention (+0.20±0.91/month, p=0.824) of knowledge. On average, nurses scored lower than physicians (-19.39±3.30, p<0.0001). Lost to follow-up had a significant impact on knowledge retention (-3.03±0.88/month, p=0.0007). CONCLUSIONS: aHIT for care of the seriously ill child significantly increased provider knowledge and loss of knowledge occurred over time. IMCI training did not significantly impact overall knowledge acquisition nor retention, while professional status impacted overall score and lost to follow-up impacted retention.

Modifications of the deep circumflex iliac artery free flap for reconstruction of the maxilla.

BACKGROUND: The deep circumflex iliac artery (DCIA) free flap remains underused in maxillectomy reconstruction. A number of surgical techniques have been described however, maxillary defects vary greatly and modifying techniques to account for such variation can be challenging. PURPOSE: This article presents the first standardized approach to DCIA free flap modification for maxillary reconstruction where graded modifications are made to a standard procedure based on defect grade. A review of 11 cases that underwent maxillectomy reconstruction with this technique is presented. METHODS: Defect complexity is stratified according to the Brown Classification System and graded modifications of increasing complexity are made to a standard harvest and flap inset technique. Modifications include increasing the depth of the harvested iliac crest bone to correspond to the height of the anterior maxillary wall defect, addition of a titanium mesh plate to reconstruct the orbital floor and harvest of the internal oblique muscle to fill the orbital cavity. Short and long-term outcomes and complications of 11 cases that underwent maxillectomy reconstruction according to this technique were documented. RESULTS: Defects ranged from Brown Class I-IV, b-c. All but two patients had malignant diagnoses with squamous cell carcinoma (n = 5) being the most prevalent. Short-term flap related complications were neck cellulitis (n = 1) and donor site haematoma (n = 2) whilst long-term flap related complications were mild trismus (n = 1) and donor site pain (n = 1). There were no reported problems with speech, swallowing or vision. CONCLUSIONS: This stepwise approach to DCIA free flap modification for maxillectomy defect reconstruction may be used as a guide for future maxillary reconstruction.

Multiple micronutrient supplementation transiently ameliorates environmental enteropathy in Malawian children aged 12-35 months in a randomized controlled clinical trial.

BACKGROUND: Environmental enteropathy (EE) is subclinical, diffuse villous atrophy characterized by T cell infiltration of the small intestinal mucosa associated with nutrient malabsorption and stunting. EE is assessed by the lactulose:mannitol (L:M) test, whereby nonmetabolized sugars are ingested and quantified in the urine. Multiple micronutrient (MN) deficiency morphologically mimics EE, and ω-3 (n-3) polyunsaturated fatty acids reduce mucosal inflammation in Crohn disease. OBJECTIVE: We tested the hypothesis that supplementary MNs, with or without fish oil (FO), would improve L:M in rural Malawian children aged 1-3 y compared with a control (C) group receiving a placebo. METHODS: The MNs and FO provided the Recommended Dietary Intake for 26 vitamins, minerals, eicosapentaenoic acid, and docosahexaenoic acid. This was a 3-arm, randomized, double-blind, placebo-controlled clinical trial, with the primary outcomes being the change in L:M (ΔL:M) after 12 and 24 wk of supplementation. Comparisons were made for ΔL:M after 12 and 24 wk within each group by using a Wilcoxon matched pairs signed rank test, because the data are not normally distributed. RESULTS: A total of 230 children had specimens adequate for analysis; all had an abnormal baseline L:M, defined as >0.10. After 12 wk, children who received MNs + FO had a ΔL:M [mean (95% CI)] of -0.10 (-0.04, -0.15; P = 0.001), and children receiving only MNs had ΔL:M of -0.12 (-0.03, -0.21; P = 0.002). After 24 wk, children who received MNs + FO had a ΔL:M of -0.09 (-0.03, -0.15; P = 0.001); children receiving only MNs had a ΔL:M of -0.11 (-0.02, -0.20; P = 0.001), and the C group had ΔL:M of -0.07 (0.02, -0.16); P = 0.002). Linear growth was similar in all groups, ∼4.3 cm over 24 wk. CONCLUSION: Although the effect was modest, these data suggest MNs can transiently ameliorate EE in rural African children. The trial was registered at clinicaltrials.gov as NCT01593033.