Next-generation sequencing-based molecular diagnosis of 82 retinitis pigmentosa probands from Northern Ireland.

Retinitis pigmentosa (RP) is a group of inherited retinal disorders characterized by progressive photoreceptor degeneration. An accurate molecular diagnosis is essential for disease characterization and clinical prognoses. A retinal capture panel that enriches 186 known retinal disease genes, including 55 known RP genes, was developed. Targeted next-generation sequencing was performed for a cohort of 82 unrelated RP cases from Northern Ireland, including 46 simplex cases and 36 familial cases. Disease-causing mutations were identified in 49 probands, including 28 simplex cases and 21 familial cases, achieving a solving rate of 60 %. In total, 65 pathogenic mutations were found, and 29 of these were novel. Interestingly, the molecular information of 12 probands was neither consistent with their initial inheritance pattern nor clinical diagnosis. Further clinical reassessment resulted in a refinement of the clinical diagnosis in 11 patients. This is the first study to apply next-generation sequencing-based, comprehensive molecular diagnoses to a large number of RP probands from Northern Ireland. Our study shows that molecular information can aid clinical diagnosis, potentially changing treatment options, current family counseling and management.

Gut microbiome homeostasis and the future of probiotics in cancer immunotherapy.

The gut microbiome has an impact on cancer immune surveillance and immunotherapy, with recent studies showing categorical differences between immunotherapy-sensitive and immunotherapy-resistant cancer patient cohorts. Although probiotics are traditionally being supplemented to promote treatments or sustain therapeutic benefits; the FDA has not approved any for use with immunotherapy. The first step in developing probiotics for immunotherapy is identifying helpful or harmful bacteria down to the strain level. The gut microbiome's heterogeneity before and during treatment is also being investigated to determine microbial strains that are important for immunotherapy. Moreover, Dietary fiber intake, prebiotic supplementation and fecal microbiota transplantation (FMT) were found to enhance intratumoral CD8+ T cell to T-reg ratio in the clinics. The possibility of probiotic immunotherapy as a "living adjuvant" to CAR treatment and checkpoint blockade resistance is actively being investigated.

Molecular diagnosis for heterogeneous genetic diseases with targeted high-throughput DNA sequencing applied to retinitis pigmentosa.

BACKGROUND: The genetic heterogeneity of many Mendelian disorders, such as retinitis pigmentosa which results from mutations in over 40 genes, is a major obstacle to obtaining a molecular diagnosis in clinical practice. Targeted high-throughput DNA sequencing offers a potential solution and was used to develop a molecular diagnostic screen for patients with retinitis pigmentosa. METHODS: A custom sequence capture array was designed to target the coding regions of all known retinitis pigmentosa genes and used to enrich these sequences from DNA samples of five patients. Enriched DNA was subjected to high-throughput sequencing singly or in pools, and sequence variants were identified by alignment of up to 10 million reads per sample to the normal reference sequence. Potential pathogenicity was assessed by functional predictions and frequency in controls. RESULTS AND CONCLUSIONS: Known homozygous PDE6B and compound heterozygous CRB1 mutations were detected in two patients. A novel homozygous missense mutation (c.2957A→T; p.N986I) in the cyclic nucleotide gated channel ß1 (CNGB1) gene predicted to have a deleterious effect and absent in 720 control chromosomes was detected in one case in which conventional genetic screening had failed to detect mutations. The detection of known and novel retinitis pigmentosa mutations in this study establishes high-throughput DNA sequencing with DNA pooling as an effective diagnostic tool for heterogeneous genetic diseases.

Development of a diagnostic genetic test for simplex and autosomal recessive retinitis pigmentosa.

PURPOSE: Retinitis pigmentosa (RP) causes hereditary blindness in adults (prevalence, approximately 1 in 4000). Each of the more than 30 causative genes identified to date are responsible for only a small percentage of cases. Genetic diagnosis via traditional methods is problematic, and a single test with a higher probability of detecting the causative mutation would be very beneficial for the clinician. The goal of this study therefore was to develop a high-throughput screen capable of detecting both known mutations and novel mutations within all genes implicated in autosomal recessive or simplex RP. DESIGN: Evaluation of diagnostic technology. PARTICIPANTS AND CONTROLS: Participants were 56 simplex and autosomal recessive RP patients, with 360 population controls unscreened for ophthalmic disease. METHODS: A custom genechip capable of resequencing all exons containing known mutations in 19 disease-associated genes was developed (RP genechip). A second, commercially available arrayed primer extension (APEX) system was used to screen 501 individual previously reported variants. The ability of these high-throughput approaches to identify pathogenic variants was assessed in a cohort of simplex and autosomal recessive RP patients. MAIN OUTCOME MEASURES: Number of mutations and potentially pathogenic variants identified. RESULTS: The RP genechip identified 44 sequence variants: 5 previously reported mutations; 22 known single nucleotide polymorphisms (SNPs); 11 novel, potentially pathogenic variants; and 6 novel SNPs. There was strong concordance with the APEX array, but only the RP genechip detected novel variants. For example, identification of a novel mutation in CRB1 revealed a patient, who also had a single previously known CRB1 mutation, to be a compound heterozygote. In some individuals, potentially pathogenic variants were discovered in more than one gene, consistent with the existence of disease modifier effects resulting from mutations at a second locus. CONCLUSIONS: The RP genechip provides the significant advantage of detecting novel variants and could be expected to detect at least one pathogenic variant in more than 50% of patients. The APEX array provides a reliable method to detect known pathogenic variants in autosomal recessive RP and simplex RP patients and is commercially available. High-throughput genotyping for RP is evolving into a clinically useful genetic diagnostic tool. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Administrative data fail to accurately identify cases of healthcare-associated infection.

OBJECTIVE: Some policy makers have embraced public reporting of healthcare-associated infections (HAIs) as a strategy for improving patient safety and reducing healthcare costs. We compared the accuracy of 2 methods of identifying cases of HAI: review of administrative data and targeted active surveillance. DESIGN, SETTING, AND PARTICIPANTS: A cross-sectional prospective study was performed during a 9-month period in 2004 at the Children's Hospital of Philadelphia, a 418-bed academic pediatric hospital. "True HAI" cases were defined as those that met the definitions of the National Nosocomial Infections Surveillance System and that were detected by a trained infection control professional on review of the medical record. We examined the sensitivity and the positive and negative predictive values of identifying HAI cases by review of administrative data and by targeted active surveillance. RESULTS: We found similar sensitivities for identification of HAI cases by review of administrative data (61%) and by targeted active surveillance (76%). However, the positive predictive value of identifying HAI cases by review of administrative data was poor (20%), whereas that of targeted active surveillance was 100%. CONCLUSIONS: The positive predictive value of identifying HAI cases by targeted active surveillance is very high. Additional investigation is needed to define the optimal detection method for institutions that provide HAI data for comparative analysis.

Mutational Analysis of the Rhodopsin Gene in Sector Retinitis Pigmentosa.

BACKGROUND: To determine the role of rhodopsin (RHO) gene mutations in patients with sector retinitis pigmentosa (RP) from Northern Ireland. DESIGN: A case series of sector RP in a tertiary ocular genetics clinic. PARTICIPANTS: Four patients with sector RP were recruited from the Royal Victoria Hospital (Belfast, Northern Ireland) and Altnagelvin Hospital (Londonderry, Northern Ireland) following informed consent. METHODS: The diagnosis of sector RP was based on clinical examination, International Society for Clinical Electrophysiology of Vision (ISCEV) standard electrophysiology, and visual field analysis. DNA was extracted from peripheral blood leucocytes and the coding regions and adjacent flanking intronic sequences of the RHO gene were polymerase chain reaction (PCR) amplified and cycle sequenced. MAIN OUTCOME MEASURE: Rhodopsin mutational status. RESULTS: A heterozygous missense mutation in RHO (c.173C > T) resulting in a non-conservative substitution of threonine to methionine (p. Thr58Met) was identified in one patient and was absent from 360 control individuals. This non-conservative substitution (p.Thr58Met) replaces a highly evolutionary conserved polar hydrophilic threonine residue with a non-polar hydrophobic methionine residue at position 58 near the cytoplasmic border of helix A of RHO. CONCLUSIONS: The study identified a RHO gene mutation (p.Thr58Met) not previously reported in RP in a patient with sector RP. These findings outline the phenotypic variability associated with RHO mutations. It has been proposed that the regional effects of RHO mutations are likely to result from interplay between mutant alleles and other genetic, epigenetic and environmental factors.

An outbreak of mediastinitis among heart transplant recipients apparently related to a change in the united network for organ sharing guidelines.

OBJECTIVE: To describe an outbreak of mediastinitis in heart transplant recipients. DESIGN: Retrospective and contemporaneous cohort SETTING: Urban tertiary-care university hospital with a large cardiac transplantation program. PATIENTS: Heart transplant recipients. INTERVENTIONS: Modifications of donor harvest technique; procedures aimed at decreasing skin and mucosal bacterial colonization; strict aseptic technique in the intensive care unit; and aggressive policing of established infection control practices. RESULTS: In April 1999, mediastinitis rates among heart transplant recipients increased abruptly from a baseline of 6 cases per 100 procedures to sequential quarterly rates of 22, 31, and 50 cases per 100 procedures, whereas infection rates in other cardiac operations were unchanged. Bacteria causing these infections were multidrug-resistant "nosocomial" organisms. The epidemic occurred 2 months after a change in the United Network for Organ Sharing organ allocation algorithm. This change resulted in an increase in the duration of preoperative hospitalization from a median of 52 to 79 days (P = .008) and may have promoted prolonged hospitalization of patients with high illness severity. Aggressive multidisciplinary interventions were temporally associated with a return to preoperative mediastinitis rates without changing length of hospitalization prior to transplantation. CONCLUSIONS: Changes in organ allocation for transplant that prolong waiting time in the hospital and alter illness acuity may lead to increased rates of postoperative infection. Measures to limit bacterial colonization may be a helpful countervailing strategy.

Infection prevention disaster preparedness planning for long-term care facilities.

BACKGROUND: Long-term care facilities (LTCFs) are defined as residential institutions that provide care to people who are unable to live independently. Planning for infection prevention in disaster situations is essential for LTCFs because of the increased risk inherent in their patient population. Experiences with past disasters, such as pandemic influenza and Hurricane Katrina, have demonstrated where LTCFs are lacking in preparedness and opportunities for improvement. Little guidance is currently available to assist these facilities in creating an infection prevention component for their disaster plans. This paper is intended to guide the development of an infection prevention component of the LTCF disaster plan. METHODS: A literature review and Internet search were conducted in September 2010. A spreadsheet was created with infection prevention topics for disaster plans that were identified. Recommendations were divided into themes/domains for simplification and clarity. RESULTS: Fifty-eight articles, planning documents/reports, and Web-based training programs were identified and screened. Of the sources screened, 33 publications were determined to be relevant; 22 of which were peer-reviewed journal articles, and 11 were state, federal, or regulatory agency publications. CONCLUSION: Whereas there were multiple publications related to the difficulties and risk factors LTCFs face in disasters, there were no publications that specifically addressed infection prevention in disasters or planning specific to infection prevention concerns in disasters in long-term care. LTCF administrators or others responsible for disaster planning in LTCFs are encouraged to use this article as a guide to developing comprehensive infection prevention policies and protocols for their emergency operations plan.

Personal protective equipment use and allocation in home health during disasters.

BACKGROUND: Home health preparedness for disasters is imperative, including the need to identify essential resources to protect home health professionals from exposure during an event. Access to personal protective equipment (PPE) is expected to be limited during a disaster, and PPE distribution and allocation needs to be prearranged to minimize infection transmission risk. This article outlines the appropriate use and allocation of PPE for home health agencies as part of disaster planning. METHODS: A literature review and Internet search were conducted in July-August 2010. A spreadsheet was created delineating the best practices related to PPE use and allocation identified by each source. Recommendations were divided into themes/domains for simplification and clarity. RESULTS: A total of 46 articles, planning documents/reports, and Web-based training programs were identified and screened. Of these, 28 were deemed relevant, including 12 journal articles and 16 published reports, book chapters, planning documents, or training programs. Themes for PPE use and allocation in home health during disasters in the literature included identifying the types of PPE used in home health, determining PPE needs, storing PPE, allocating PPE when resources are limited or depleted, disposing of PPEl, and educating staff. CONCLUSION: Having access to the correct types and quantities of PPE during a disaster will be essential to home health agencies. The information presented in this article can assist home health agencies in developing emergency management plans that address planning issues related to PPE.