Utility of next-generation sequencing technologies for the efficient genetic resolution of haematological disorders.

Next-generation sequencing (NGS) has now evolved to be a relatively affordable and efficient means of detecting genetic mutations. Whole genome sequencing (WGS) or whole exome sequencing (WES) offers the opportunity for rapid diagnosis in many paediatric haematological conditions, where phenotypes are variable and either a large number of genes are involved, or the genes are large making sanger sequencing expensive and labour-intensive. NGS offers the potential for gene discovery in patients who do not have mutations in currently known genes. This report shows how WES was used in the diagnosis of six paediatric haematology cases. In four cases (Diamond-Blackfan anaemia, congenital neutropenia (n = 2), and Fanconi anaemia), the diagnosis was suspected based on classical phenotype, and NGS confirmed those suspicions. Mutations in RPS19, ELANE and FANCD2 were found. The final two cases (MYH9 associated macrothrombocytopenia associated with multiple congenital anomalies; atypical juvenile myelomonocytic leukaemia associated with a KRAS mutation) highlight the utility of NGS where the diagnosis is less certain, or where there is an unusual phenotype. We discuss the advantages and limitations of NGS in the setting of these cases, and in haematological conditions more broadly, and discuss where NGS is most efficiently used.

"Choosing Wisely Canada" and antimicrobial stewardship: A shared focus on reducing unnecessary care.

There is a growing movement in medicine which recognizes that some tests, treatments or procedures do not add value for patients, and may even cause harm. The "Choosing Wisely Canada" campaign is a grassroots, physician-led campaign to engage physicians and patients in conversations about overuse of unnecessary tests, treatments and procedures to improve the quality of health care. This article reviews the underlying principles of this campaign and its spread across Canada. It also highlights the alignment between the principles of Choosing Wisely Canada with those of antimicrobial stewardship, which share similar motivations, challenges and opportunities.

MeCP2 deficiency is associated with reduced levels of tubulin acetylation and can be restored using HDAC6 inhibitors.

UNLABELLED: Rett syndrome (RTT) is a severe neurodevelopmental disorder, predominantly caused by loss of function mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene. Despite the genetic cause being known in the majority of cases, the pathophysiology of the neurological phenotype of RTT is largely unknown. Tubulin and the microtubule network play an essential role in neuronal function whereby the acetylation state of microtubules dictates the efficiency of neuronal migration and differentiation, synaptic targeting and molecular motor trafficking of mRNA, high-energy mitochondria and brain-derived neurotrophic factor (BDNF)-containing vesicles. Recent reports have shown perturbations in tubulin and microtubule dynamics in MeCP2-deficient cells, suggesting a link between the aberrations of these cellular entities and the neurobiology of RTT. We have interrogated the functional state of the microtubule network in fibroblasts derived from two patients with RTT as well as cortical neurons from a RTT mouse model and observed a reduction in acetylated α-tubulin and an increase in the tubulin-specific deacetylase, histone deacetylase 6 (HDAC6). Furthermore, we show that inhibition of HDAC6 by Tubastatin A can restore tubulin acetylation levels. We also demonstrate microtubule instability in the RTT patient fibroblasts in response to nocodazole, which is progressively ameliorated in a mutation-dependent manner by Tubastatin A. We conclude that Tubastatin A is capable of counteracting the microtubule defects observed in MeCP2-deficient cells, which could in turn lead to the restoration of molecular trafficking along the microtubules and thus could be a potentially new therapeutic option for RTT. KEY MESSAGE: Cells from MeCP2-deficient cells show reduced levels of acetylated α-tubulin. Cells from two patients and a RTT mouse model have increased levels of HDAC6 but not sirtuin 2 (SIRT2). Inhibition of HDAC6 by Tubastatin A increases the in vitro acetylation of α-tubulin. Inhibition of HDAC6 by Tubastatin A does not increase MECP2 expression. Cells from two patients show microtubule instability, which is ameliorated by Tubastatin A.

Mitochondrial dysfunction in the skeletal muscle of a mouse model of Rett syndrome (RTT): implications for the disease phenotype.

Rett syndrome (RTT) is a severe neurodevelopmental disorder, predominantly caused by mutations in the X-linked Methyl-CpG-binding protein 2 (MECP2) gene. Patients present with numerous functional deficits including intellectual disability and abnormalities of movement. Clinical and biochemical features may overlap with those seen in patients with primary mitochondrial respiratory chain disorders. In the late stages of the disorder, patients suffer from motor deterioration and usually require assisted mobility. Using a mouse model of RTT (Mecp2(tm1Tam)), we studied the mitochondrial function in the hind-limb skeletal muscle of these mice. We identified a reduction in cytochrome c oxidase subunit I (MTCO1) at both the transcript and protein level, in accordance with our previous findings in RTT patient brain studies. Mitochondrial respiratory chain (MRC) enzyme activity of complexes II+III (COII+III) and complex IV (COIV), and glutathione (GSH) levels were significantly reduced in symptomatic mice, but not in the pre-symptomatic mice. Our findings suggest that mitochondrial abnormalities in the skeletal muscle may contribute to the progressive deterioration in mobility in RTT through the accumulation of free radicals, as evidenced by the decrease in reduced glutathione (GSH). We hypothesise that a diminution in GSH leads to an accumulation of free radicals and an increase in oxidative stress. This may impact on respiratory chain function and contribute in part to the progressive neurological and motor deterioration seen in the Mecp2-mutant mouse. Treatment strategies aimed at restoring cellular GSH levels may prove to be a novel target area to consider in future approaches to RTT therapies.

Understanding, compliance and psychological impact of the SARS quarantine experience.

This study examines a cohort of persons quarantined during the 2003 SARS outbreak in Canada and describes their understanding of, difficulties and compliance with, and the psychological impact of the quarantine experience. A mailed questionnaire was administered to 1912 eligible adults and included the Impact of Events Scale - Revised (IES-R) to assess symptoms of post-traumatic stress disorder (PTSD). Self-reported compliance with all required quarantine measures was low (15.8+/-2.3%), although significantly higher when the rationale for quarantine was understood (P=0.018). Health-care workers (HCW) experienced greater psychological distress, including symptoms of PTSD (P<0.001). Increasing perceived difficulty with compliance, HCW, longer quarantine and compliance with quarantine requirements were significant contributors to higher IES-R scores. The low compliance with quarantine requirements introduces concerns about the effectiveness of quarantine as a public health measure. Improvements in compliance and reduced psychological distress may be possible by minimizing duration, revising requirements, and providing enhanced education and support.

Chronic dyspnea and hyperventilation in an awake patient with small subcortical infarcts.

A 79-year-old woman presented with chronic dyspnea and hyperventilation. There was no evidence of pulmonary disease. Hyperventilation persisted during sleep and after high-dose administration of a narcotic. A head MRI revealed bilateral medial thalamic infarctions. Central neurogenic hyperventilation was diagnosed in this alert patient. The case may illustrate a role for the thalamus in regulating ventilation, but another small infarct not visible on MRI also could be responsible.

Thrombogenicity of radiofrequency ablation procedures: what factors influence thrombin generation?

AIMS: Thromboembolic complications have been reported after radiofrequency ablation but the low incidence of overt clinical events has been a limitation to the study of factors affecting thrombogenic risk. The aim of this study was to determine whether radiofrequency ablation has a procoagulant effect and to examine variables that affect thrombio generation. METHODS AND RESULTS: Thirty-seven consecutive patients who underwent radiofrequency ablation were studied prospectively. Blood samples were assayed for thrombin-antithrombin III (TAT) and d-dimer (DD) at five different time points: (1) baseline; (2) after sheath insertion; (3) after electrophysiological study but before radiofrequency ablation; (4) at completion of the procedure; and (5) 24 h post-procedure. TAT levels were within the normal range at baseline and increased significantly after sheath insertion from 2.1 +/- 1.2 microg l(-1) to 13.3 +/- 16.0 microg l(-1) (P<0.01). Levels increased further to 24.0 +/- 19.9 microg l(-1) (P<0.01) after electrophysiological study but did not increase after radiofrequency ablation. TAT normalized at 24 h. DD increased significantly from baseline values (230.2 +/- 176.8 ng ml(-1)) to 285.4 +/- 237.4 ng ml(-1) (P=0.019) after sheath insertion. There was a further significant increase after electrophysiological study to 423.4 +/- 324.3 ng ml(-1) (P<0.01), and a slight but non-significant increase to 464.4 +/- 307.4 ng ml(-1) after radiofrequency ablation (P=0.159). DD remained elevated at 24 h. Procedure duration was the only variable that correlated with the relative increase in TAT and DD. The patients with the longest procedure durations had more catheters inserted, more radiofrequency applications and largely consisted of accessory bypass tract-mediated tachycardias. Heparin administration significantly blunted the relative increase in TAT after radiofrequency ablation (P=0.005). CONCLUSION: Radiofrequency ablation procedures confer an increased risk of thrombosis. Catheterization and diagnostic study contribute largely to the thrombogenic stimulus. Thrombogenicity is increased in prolonged, complex procedures and is decreased in patients who have been administered heparin during the procedure.

Determinants of skeletal muscle catabolism after severe burn.

OBJECTIVE: To determine which patient factors affect the degree of catabolism after severe burn. SUMMARY BACKGROUND DATA: Catabolism is associated with severe burn and leads to erosion of lean mass, impaired wound healing, and delayed rehabilitation. METHODS: From 1996 to 1999, 151 stable-isotope protein kinetic studies were performed in 102 pediatric and 21 adult subjects burned over 20-99. 5% of their total body surface area (TBSA). Patient demographics, burn characteristics, and hospital course variables were correlated with the net balance of skeletal muscle protein synthesis and breakdown across the leg. Data were analyzed sequentially and cumulatively through univariate and cross-sectional multiple regression. RESULTS: Increasing age, weight, and delay in definitive surgical treatment predict increased catabolism (P < .05). Body surface area burned increased catabolism until 40% TBSA was reached; catabolism did not consistently increase thereafter. Resting energy expenditure and sepsis were also strong predictors of net protein catabolism. Among factors that did not significantly correlate were burn type, pneumonia, wound contamination, and time after burn. From these results, the authors also infer that gross muscle mass correlates independently with protein wasting after burn. CONCLUSIONS: Heavier, more muscular subjects, and subjects whose definitive surgical treatment is delayed are at the greatest risk for excess catabolism after burn. Sepsis and excessive hypermetabolism are also associated with protein catabolism.

The effects of medical group practice and physician payment methods on costs of care.

OBJECTIVE: To assess the effects of payment methods on the costs of care in medical group practices. DATA SOURCES: Eighty-six clinics providing services for a Blue Cross managed care program during 1995. The clinics were analyzed to determine the relationship between payment methods and cost of care. Cost and patient data were obtained from Blue Cross records, and medical group practice clinic data were obtained by a survey of those organizations. STUDY DESIGN: The effects of clinic and physician payment methods on per member per year (PMPY) adjusted patient costs are evaluated using a two-stage regression model. Patient costs are adjusted for differences in payment schedules; patient age, gender, and ACG; clinic organizational variables are included as explanatory variables. DATA COLLECTION: Patient cost data were extracted from Blue Cross claims files, and patient and physician data from their enrollee and provider data banks. Medical group practice data were obtained by a mailed survey with telephone follow-up. PRINCIPAL FINDINGS: Capitation payment is correlated with lower patient care costs. When combined with fee-for-service with withhold provisions, this effect is smaller indicating that these two clinic payment methods are not interchangeable. Clinics with more physician compensation based on measures of resource use or based on some share of the net revenue of the clinic have lower patient care costs than those with more compensation related to productivity or based on salary. Salary compensation is strongly associated with higher costs. The use of physician profiles and clinical guidelines is associated with lower costs, but referral management systems have no such effect. The lower cost clinics are the smaller, multispecialty clinics. CONCLUSIONS: This study indicates that payment methods at both the medical group practice and physician levels influence the cost of care. However, the methods by which that influence is manifest is not clear. Although the organizational structure of clinics and their use of managed care programs appear to play a role, this influence is less than expected.

Ethnic differences: word descriptors used by African-American and white asthma patients during induced bronchoconstriction.

STUDY OBJECTIVES: To determine if African-American and white patients with asthma (1) differ in the words they use to describe their breathlessness, and (2) differ in their perception of breathlessness. DESIGN: Descriptive cross-sectional design. SETTING AND PARTICIPANTS: The study setting was located in Northern California, an ethnically and economically diverse area. A total of 32 subjects, 16 per group, completed the study. MEASUREMENTS: All had a provocation concentration of methacholine chloride causing a 30% fall in FEV(1) (PC(30)) of

Measurement of fibroblast proliferative activity in bronchoalveolar lavage fluid in the analysis of obliterative bronchiolitis among lung transplant recipients.

BACKGROUND: Bronchiolitis obliterans occurs in 30% to 80% of lung-transplant recipients and is a direct cause of death in more than 40% of patients with this complication. This study assessed the potential utility of measuring fibroblast-proliferative activity in bronchoalveolar lavage fluid from lung-transplant recipients to better understand the pathogenesis of this process. METHODS: The capacity of bronchoalveolar lavage fluid obtained from transplant recipients, during routine surveillance bronchoscopy, to stimulate the proliferation of human lung fibroblasts in vitro was assessed retrospectively and compared to that of control subjects. For each recipient, a correlation was made between the fibroblast-proliferative activity in serial lavage samples over time and the other modalities employed for detecting post-transplant complications including spirometry, transbronchial lung biopsy, and high-resolution computed tomography. RESULTS: There was a significant difference in fibroblast-proliferative activity between volunteer and transplant recipient groups (p = 0.002). Further, for each transplant recipient, the decline in the forced expired flow rate between 25% and 75% of expired volume (FEF(25%-75%)) was correlated with the mean fibroblast-proliferative activity during the period of this study (r = 0.83; p = 0.04). CONCLUSIONS: A sustained increase in fibroblast-proliferative activity in lavage supernatant precedes both histologic and physiologic evidence of bronchiolitis obliterans. Relative to an increase in fibroblast-proliferative activity or abnormalities in FEF25%-75%, a decrease in forced expiratory volume in 1 second is a late finding.

Pulmonary function improves after expandable metal stent placement for benign airway obstruction.

STUDY OBJECTIVE: To determine whether expandable metal stent placement for benign airway lesions improves pulmonary function. DESIGN: Case series. SETTING: University medical center. PATIENTS: Nine patients who underwent balloon-mediated expandable metal stent deployment for airway obstruction due to benign etiologies. RESULTS: All nine patients had expandable stents deployed for benign airway lesions using fiberoptic bronchoscopy and fluoroscopic guidance. Pulmonary function improved after stent placement. The mean FVC increased by 388 mL (95% confidence interval [CI], 30 to 740 mL), the mean peak expiratory flow (PEF) increased by 1,288 mL (95% CI, 730 to 1,840 mL), the mean FEV1 increased by 550 mL (95% CI, 240 to 860 mL), and the mean forced expiratory flow between 25% and 50% of vital capacity (FEF25-75%) increased by 600 mL (95% CI, 110 to 1,090 mL). Corresponding relative measurements included increases in FVC (12%), PEF (95%), FEV1 (38%), and FEF25-75% (87%). The complete characterization of a benign airway obstruction generally required a multimodal approach. CONCLUSIONS: Expandable metal stent placement appears to be an effective therapy for benign airway obstruction.

Intramedullary abscess of the spinal cord in the antibiotic era: clinical features, microbial etiologies, trends in pathogenesis, and outcomes.

We review the clinical features, microbial etiologies, mechanisms of infection, and outcomes of 25 cases of intramedullary abscess of the spinal cord (IASC) reported between 1977 and 1997, the modern era. All patients presented with motor and/or sensory neurological deficits. Back pain and/or radicular pain was common (60%); fever was present in a minority (40%) of patients. Preexisting abnormalities of the spinal cord and/or vertebral column were present in 44% of cases. Contiguous spread of infection through a congenital dermal sinus was the mechanism of infection in 24% of cases. The infection was fatal in 8% of cases; persistent neurological deficits were documented in 70% of patients who survived. To assess the impact of antimicrobial therapy on the pathogenesis and outcomes of IASC, cases reported in the modern era are compared with 42 cases of IASC reported between 1830 and 1944, the preantibiotic era.

Pyogenic brain abscess caused by Streptococcus pneumoniae: case report and review.

While Streptococcus pneumoniae is the most common cause of bacterial meningitis in adults, cases of pneumococcal brain abscess have rarely been reported. We describe a case of otogenic brain abscess caused by S. pneumoniae that developed in a patient who was receiving ciprofloxacin for the empirical treatment of otitis media. We also review 23 additional cases of pyogenic brain abscess caused by S. pneumoniae that have previously been reported. The development of a pneumococcal brain abscess was associated with a contiguous intracranial focus of infection in 50% of cases. The majority of patients presented with headache (81%) and focal neurological deficits (86%). However, the classic triad of headache, fever, and focal neurological deficits was present in only 24% of patients. The mortality rate for patients with brain abscess caused by S. pneumoniae was 35%; persistent neurological deficits were documented in 40% of patients who survived.

Delayed diagnosis of acute rheumatic fever in adults: a forgotten cause of febrile polyarthritis.

Acute rheumatic fever is a nonsuppurative sequela of upper respiratory tract infection with group A streptococci. We describe our recent experience with the diagnosis and management of 3 cases of acute rheumatic fever to highlight the delays that may arise in the diagnosis of this condition. In adults, febrile polyarthritis is the most common presentation of acute rheumatic fever. Increased awareness on the part of the physician is necessary to ensure both prompt and accurate diagnosis of this cause of febrile polyarthritis.

Eye drops--the hidden poison.

Eye drops are very commonly prescribed but their potential for systemic absorption and serious toxicity may be forgotten. This paper examines patterns of prescription of eye drops in Scotland by general practitioners. A review of the serious systemic features and toxicity, and their management, of commonly used eye drops is undertaken. Practical recommendations for the monitoring of such effects in clinical practice are made.

Can ultrasound probes and coupling gel be a source of nosocomial infection in patients undergoing sonography? An in vivo and in vitro study.

OBJECTIVE: At our institution, ultrasound probes are wiped with a clean, dry, soft, absorbent paper towel after each procedure as a basic standard of probe disinfection. However, it was unclear if this provided a sufficient level of decontamination. This study was designed to determine if the ultrasound probe and coupling gel can act as a vector of nosocomial infection and to describe a cost-effective method of probe handling that allows optimal control of infection. SUBJECTS AND METHODS: In the first part of the study, the ultrasound probe was exposed to the disrupted skin of patients recruited from our inpatient population, using our routine scanning technique to look for subcutaneous collections. Twenty-seven patients were scanned: 17 with surgical wounds, seven with surgical drains, four with enteric stomas, three with biopsy sites, and three with ulcers or excoriation. Fifteen patients had a discharge associated with their disrupted skin, and seven patients had culture-proved skin infections. Each probe was wiped with a clean, dry paper towel after scanning, then immersed in a brain-heart infusion (BHI) broth, and the solution was cultured. In the second part of the study, the ultrasound probe was exposed to a large inoculum of bacteria. Sixty-one probes were used to scan fields of confluent growth of bacteria on agar plates. Twenty-six probes were cleaned by wiping with a dry, clean paper towel, and 25 probes were cleaned by wiping with a dry, clean paper towel followed by immersion in Hibidil (0.05% chlorhexidine weight/volume). Ten probes functioned as controls and were not cleaned after exposure to the bacteria. Each probe was then immersed in BHI broth, and the solution was cultured. In the third part of the study, the coupling gel was evaluated as a culture medium for bacterial growth. Twenty-five agar plates were inoculated with a confluent growth of bacteria. Half of the surface of each agar plate was covered with coupling gel, and the remaining surface was left unexposed. The resulting bacterial growth on each side of the plates was compared. RESULTS: One of the 27 probes exposed to patients with disrupted skin grew Staphylococcus epidermidis (skin flora). For probes exposed to a large inoculum of bacteria, we found no statistically significant difference in the number of probes that showed bacterial growth on culture between probes cleaned by wiping with a towel and those cleaned with Hibidil. Furthermore, the resulting bacterial growth in both sets of probes was scant and was not considered clinically significant. All 10 control probes showed clinically significant growth in all cases. As for evaluation of the coupling gel as a culture medium, the gel permitted bacterial growth and did not show any evidence of bacteriocidal or bacteriostatic properties. CONCLUSION: Ultrasound probes that are wiped with a paper towel until they are visibly clean do not contribute to nosocomial infections. Additional antiseptic solutions such as Hibidil are not necessary. We suggest that probes be simply wiped with a clean, dry, nonsterile paper towel between procedures, including probes used on contaminated scanning fields, open wounds, and cutaneous infections. After the final procedure of the day, probes should be cleaned with a liquid cleaning solution such as Hibidil to remove all traces of coupling gel, which could support the overnight growth of bacteria. This would decontaminate the probes and prevent the overnight growth of bacteria. This method would be both a cost-effective and time-efficient protocol for controlling infection.