Rebecca B. Saunderson

Denaturing high performance liquid chromatography: high throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease

Aims: To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) as a high throughput tool in: (1) DNA mutation detection in familial hypertrophic cardiomyopathy (FHC), and (2) single nucleotide polymorphism (SNP) discovery and validation in sporadic motor neurone disease (MND). Methods: The coding sequence and intron–exon boundaries of the cardiac β myosin heavy chain gene (MYH7) were screened by DHPLC for mutation identification in 150 unrelated patients diagnosed with FHC. One hundred and forty patients with sporadic MND were genotyped for the A67T SNP in the poliovirus receptor gene. All DHPLC positive signals were confirmed by conventional methods. Results: Mutation screening of MYH7 covered 10 kb with a total of 5700 amplicons, and more than 6750 DHPLC injections were completed within 35 days. The causative mutation was identified in 14% of FHC cases, including seven novel missense mutations (L227V, E328G, K351E, V411I, M435T, E894G, and E927K). Genotyping of the A67T SNP was performed at two different temperatures both in MND cases and 280 controls. This coding SNP was found more frequently in MND cases (13.6%) than in controls (6.8%). Furthermore, 19 and two SNPs were identified in MYH7 and the poliovirus receptor gene, respectively, during DHPLC screening. Conclusions: DHPLC is a high throughput, sensitive, specific, and robust platform for the detection of DNA variants, such as disease causing mutations or SNPs. It enables rapid and accurate screening of large genomic regions.

Impact of routine bedside infectious disease consultation on clinical management and outcome of Staphylococcus aureus bacteraemia in adults

Staphylococcus aureus bacteraemia (SAB) is a common, serious infection that is associated with high rates of morbidity and mortality. Evidence suggests that infectious disease consultation (IDC) improves clinical management in patients with SAB. We examined whether the introduction of a routine bedside IDC service for adults with SAB improved clinical management and outcomes compared to telephone consultation. We conducted an observational cohort study of 571 adults with SAB at a teaching hospital in the United Kingdom between July 2006 and December 2012. A telephone consultation was provided on the day of positive blood culture in all cases, but an additional bedside IDC was provided after November 2009 (routine IDC group). Compared to patients in the pre-IDC group, those in the routine IDC group were more likely to have a removable focus of infection identified, echocardiography performed and follow-up blood cultures performed. They also received longer courses of antimicrobial therapy, were more likely to receive combination antimicrobial therapy and were more likely to have SAB recorded in the hospital discharge summary. There was a trend towards lower mortality at 30 days in the routine IDC group compared to the pre-IDC group (12% vs. 22%, p 0.07). Our findings suggest that routine bedside IDC should become the standard of care for adults with SAB.

A polymorphism in the poliovirus receptor gene differs in motor neuron disease

Poliovirus has been implicated in the etiology of sporadic motor neuron disease. DNA polymorphisms in the poliovirus receptor gene (PVR) are associated with persistent poliovirus infection in cell culture. PVR DNA polymorphisms were therefore studied in 110 cases of amyotrophic lateral sclerosis, 30 cases of progressive muscular atrophy (a disorder of lower motor neurons) and 280 normal controls. In exon 2 of PVR the heterozygous Ala67Thr change was detected in 20.0% of progressive muscular atrophy, 11.8% of amyotrophic lateral sclerosis and 6.8% of control subjects. The frequency of the polymorphism was significantly higher in patients with progressive muscular atrophy than in controls. Differences in the poliovirus receptor gene may result in slowly progressive viral cytopathic effects that lead to lower motor neuron forms of motor neuron disease.

Clonal differences in Staphylococcus aureus bacteraemia-associated mortality

The bacterium Staphylococcus aureus is a major human pathogen for which the emergence of antibiotic resistance is a global public health concern. Infection severity, and in particular bacteraemia-associated mortality, has been attributed to several host-related factors, such as age and the presence of comorbidities. The role of the bacterium in infection severity is less well understood, as it is complicated by the multifaceted nature of bacterial virulence, which has so far prevented a robust mapping between genotype, phenotype and infection outcome. To investigate the role of bacterial factors in contributing to bacteraemia-associated mortality, we phenotyped a collection of sequenced clinical S. aureus isolates from patients with bloodstream infections, representing two globally important clonal types, CC22 and CC30. By adopting a genome-wide association study approach we identified and functionally verified several genetic loci that affect the expression of cytolytic toxicity and biofilm formation. By analysing the pooled data comprising bacterial genotype and phenotype together with clinical metadata within a machine-learning framework, we found significant clonal differences in the determinants most predictive of poor infection outcome. Whereas elevated cytolytic toxicity in combination with low levels of biofilm formation was predictive of an increased risk of mortality in infections by strains of a CC22 background, these virulence-specific factors had little influence on mortality rates associated with CC30 infections. Our results therefore suggest that different clones may have adopted different strategies to overcome host responses and cause severe pathology. Our study further demonstrates the use of a combined genomics and data analytic approach to enhance our understanding of bacterial pathogenesis at the individual level, which will be an important step towards personalized medicine and infectious disease management.A genome-wide association approach identifies differential biofilm and virulence attributes associated with mortality in two Staphylococcus aureus clonal complexes.

Genetic variants in the promoter of TARDBP in sporadic amyotrophic lateral sclerosis

All patients with sporadic amyotrophic lateral sclerosis (SALS) have TDP-43 inclusions in their motor neurons, suggesting this protein plays a major role in the disease. Coding mutations in the gene for TDP-43, TARDBP, have been found in only a few patients with SALS. However, the non-coding regulatory regions of TARDBP have not yet been examined in SALS. We therefore sequenced both coding and non-coding regions of TARDBP in 46 tissue-banked SALS brains (brain DNA was used to detect somatic mutations). Non-coding variants (in the promoter or intron 1) were detected in 16 patients (35%) and coding variants in 4 (9%). Two known promoter variants were found more frequently in SALS patients than in controls. Two other variants, found in one patient each but not in controls, have potential regulatory functions. In addition, a novel exon 2 change with predicted functional effects was found in one patient. In summary, variants in the promoter and other non-coding regions of TARDBP may disturb the regulation of this gene in some patients with SALS.

Mesiotemporal changes on magnetic resonance imaging in neurosyphilis.

We report a case of neurosyphilis with magnetic resonance imaging (MRI) brain scan findings compatible with a diagnosis of herpes simplex encephalitis with negative testing for herpes simplex virus in the cerebral spinal fluid. An extensive review of the literature has been undertaken revealing 24 cases worldwide where there are mesiotemporal changes on MRI concurrent with a diagnosis of neurosyphilis. Therefore, it is now well established that neurosyphilis, ‘the great imitator’, should be considered in the differential diagnosis in all patients demonstrating mesiotemporal changes on MRI, changes usually seen in herpes simplex encephalitis.

Impact of infectious diseases consultation on the management of Staphylococcus aureus bacteraemia in children

Objectives Infectious diseases consultation (IDC) in adults with Staphylococcus aureus bacteraemia (SAB) has been shown to improve management and outcome. The aim of this study was to evaluate the impact of IDC on the management of SAB in children. Study design Observational cohort study of children with SAB. Setting Cambridge University Hospitals National Health Service (NHS) Foundation Trust, a large acute NHS Trust in the UK. Participants All children with SAB admitted to the Cambridge University Hospitals NHS Foundation Trust between 16 July 2006 and 31 December 2012. Methods Children with SAB between 2006 and 31 October 2009 were managed by routine clinical care (pre-IDC group) and data were collected retrospectively by case notes review. An IDC service for SAB was introduced in November 2009. All children with SAB were reviewed regularly and data were collected prospectively (IDC group) until 31 December 2012. Baseline characteristics, quality metrics and outcome were compared between the pre-IDC group and IDC group. Results There were 66 episodes of SAB in 63 children—28 patients (30 episodes) in the pre-IDC group, and 35 patients (36 episodes) in the IDC group. The median age was 3.4 years (IQR 0.2–10.7 years). Patients in the IDC group were more likely to have echocardiography performed, a removable focus of infection identified and to receive a longer course of intravenous antimicrobial therapy. There were no differences in total duration of antibiotic therapy, duration of hospital admission or outcome at 30 or 90 days following onset of SAB. Conclusions IDC resulted in improvements in the investigation and management of SAB in children.

A comparison of the lengths of androgen receptor triplet repeats in brain and blood in motor neuron diseases

BACKGROUND Expansions of triplet repeats are found in a number of neurodegenerative conditions, and different tissues in the same person can have varying repeat lengths. In Kennedy disease, motor neuron loss is due to expansion of the CAG repeat length in the androgen receptor gene (AR). We hypothesised that patients with other sporadic motor neuron diseases could have AR expansions that were restricted to CNS tissue. METHODS We measured the AR triplet repeat length in DNA extracted from the brains of 23 patients with sporadic amyotrophic lateral sclerosis (SALS) and 3 with sporadic progressive muscular atrophy (SPMA). Paired blood samples were available in 15 patients to look for blood-brain differences in CAG repeat length. RESULTS No CAG expansions in the Kennedy disease range were found in the SALS or SPMA brains. Furthermore, no brain-blood differences were found in the lengths of AR triplet repeats. Brain AR repeat length was not associated with the duration, or age or site of onset, of disease. CONCLUSIONS The findings indicate that a brain-specific expansion of AR triplet repeats is unlikely to underlie motor neuron loss in SALS or SPMA.

Low yield in screening patients with sporadic motor neuron disease for Kennedy disease

The diagnostic yield of testing for Kennedy disease in patients diagnosed with sporadic motor neuron disease (MND) is unclear. We measured the CAG repeat lengths in the androgen receptor gene of patients with progressive limb weakness who had either upper and lower motor signs (n = 130), or lower motor neuron signs alone (n = 30). Only one patient with a long history of lower motor weakness had a repeat length in the Kennedy disease range. Testing for Kennedy disease is unlikely to benefit MND patients with upper motor neuron signs or those with a short history of lower motor signs.

Perianal Herpes Simplex Virus Infection Misdiagnosed With Pyoderma Gangrenosum: Case of the Month from the Case Consultation Committee of the International Society for the Study of Vulvovaginal Disease.

CASE REPORT A 71-year-old woman developed a small area of painful ulceration in the perianal region, which expanded in size for a 4-week period. Her relevant medical history included polymyositis, which was treated with 10 mg of methotrexate weekly, and type 2 diabetes mellitus. She denied a history of herpes labialis or genital herpes. Initially, she was reviewed by a dermatologist in the community who made a clinical diagnosis of pyoderma gangrenosum (PG) and performed a skin biopsy that was reported as consistent with PG. She was commenced on 50 mg of prednisone daily. The ulceration expanded, and 1 month later, azathioprine 1 mg/kg was added. When the lesion continued to increase in size, the patient experienced significant pain upon defecation and urination, and she was referred for assessment and management at a tertiary hospital. On physical examination, she had a large area of perianal ulceration (19 13 cm) extending to the vulva (see Figure 1). Patient consent has been obtained. The border was erythematous, was not undermined, and had some slough. There were no vesicular lesions. There was no inguinal lymphadenopathy. Per rectal examination was normal. Systems examination was unremarkable. A repeat skin biopsy showed herpes virus infection (see Figures 2A, B). Herpes simplex virus types 1 and 2 were positive on immunostain. Herpes simplex virus type 1 polymerase chain reaction was positive. The original biopsy, on review, also showed HSV infection. Her immunosuppression was ceased and she was commenced on intravenous acyclovir. She responded to treatment and re-epithelialized for a 4-week period (see Figure 3).