Clinical features, microbiology and surgical outcomes of infective endocarditis: a 13-year study from a UK tertiary cardiothoracic referral centre.

BACKGROUND: Infective endocarditis (IE) causes substantial morbidity and mortality. Patient and pathogen profiles, as well as microbiological and operative strategies, continue to evolve. The impact of these changes requires evaluation to inform optimum management and identify individuals at high risk of early mortality. AIM: Identification of clinical and microbiological features, and surgical outcomes, among patients presenting to a UK tertiary cardiothoracic centre for surgical management of IE between 1998 and 2010. DESIGN: Retrospective observational cohort study. METHODS: Clinical, biochemical, microbiological and echocardiographic data were identified from clinical records. Principal outcomes were all-cause 28-day mortality and duration of post-operative admission. RESULTS: Patients (n = 336) were predominantly male (75.0%); median age 52 years (IQR = 41-67). Most cases involved the aortic (56.0%) or mitral (53.9%) valves. Microbiological diagnoses, obtained in 288 (85.7%) patients, included streptococci (45.2%); staphylococci (34.5%); Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, Kingella (HACEK) organisms (3.0%); and fungi (1.8%); 11.3% had polymicrobial infection. Valve replacement in 308 (91.7%) patients included mechanical prostheses (69.8%), xenografts (24.0%) and homografts (6.2%). Early mortality was 12.2%, but fell progressively during the study (P = 0.02), as did median duration of post-operative admission (33.5 to 10.5 days; P = 0.0003). Multivariable analysis showed previous cardiothoracic surgery (OR = 3.85, P = 0.03), neutrophil count (OR = 2.27, P = 0.05), albumin (OR = 0.94, P = 0.04) and urea (OR = 2.63, P < 0.001) predicted early mortality. CONCLUSIONS: This study demonstrates reduced post-operative early mortality and duration of hospital admission for IE patients over the past 13 years. Biomarkers (previous cardiothoracic surgery, neutrophil count, albumin and urea), predictive of early post-operative mortality, require prospective evaluation to refine algorithms, further improve outcomes and reduce healthcare costs associated with IE.

Seasonal variation in mortality of Pneumocystis jirovecii pneumonia in HIV-infected patients.

A seasonal variation in the presentation of Pneumocystis jirovecii pneumonia (PCP) has been reported and a previous study from this centre noted a seasonal variation in mortality rates. This study examined seasonal influences (including climatic factors) within-host factors (clinical and laboratory-derived variables), the infectious burden of P. jirovecii in bronchoalveolar lavage (BAL) fluid, the presence of dihydropteroate synthase (DHPS) mutations in P. jirovecii, variations in knowledge and skills of junior medical staff, and mortality in 547 episodes of PCP occurring in 494 HIV-infected patients. The overall mortality rate was 13.5%. There was a seasonal variation in mortality: highest in autumn (21.2%) and lowest in spring (9.7%), P = 0.047. After adjustment was made for prognostic factors previously identified as being associated with mortality (increasing patient age, second/third episode of PCP, low haemoglobin, low PaO(2), presence of medical co-morbidity and pulmonary Kaposi sarcoma), there was no seasonal association with mortality, P = 0.249. The quantity of P. jirovecii DNA in BAL fluid showed no evidence of seasonal variation, P = 0.67; DHPS mutations were identified with equal frequency in each season and the mortality rate for February and August (when junior medical staff arrive in new posts) was 16.7%, only slightly greater than for other months (13.0%).

Trials and tribulations of an African-led research and capacity development programme: the case for EDCTP investments.

We describe the initiation and establishment of The University of Zambia - University College London Medical School (UNZA-UCLMS) Research and Training Project, an entirely African scientist-led, south-north partnership. In its 16 year existence, the project, by successfully obtaining competitive grant funding, has transformed itself into one of Africa's most productive African-led R&D programmes with training and visible research outputs. The project serves as a role model and now networks R&D and training activities with six southern African (10 institutions) and six European countries. This project case study illustrates that deep commitment is essential for success and that the factors which facilitate success in R&D in Africa need to be evaluated. The long-term prospects for sustaining the UNZA-UCLMS Project appear bright and are dependent on several factors: the ability to retain trained African scientists; obtaining continued competitive or donor grant funding support; and serious investment by the African governments involved. The recent 255 million Euros EDCTP investment in sub-Saharan Africa through south-north partnerships is expected to enhance existing African-led R&D programmes. African governments and scientists must rise to the challenge.

Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients.

BACKGROUND: Pneumocystis pneumonia (PCP) is conventionally diagnosed by identifying Pneumocystis jirovecii in lower respiratory tract samples using cytochemical stains. Molecular diagnosis of PCP is potentially more sensitive. METHODS: A study was undertaken to use an extensively optimised real-time polymerase chain reaction (PCR) using primers designed to hybridise with the P. jirovecii heat shock protein 70 (HSP70) gene to quantify P. jirovecii DNA in bronchoalveolar lavage (BAL) fluid from HIV-infected patients with and without PCP, and to compare this assay with conventional PCR targeting the P. jirovecii mitochondrial large subunit rRNA gene sequence (mt LSU rRNA). RESULTS: Sixty-one patients had 62 episodes of PCP (defined by detection of P. jirovecii in BAL fluid by cytochemical stains and typical clinical presentation). Quantifiable HSP70 DNA was detected in 61/62 (range approximately 13-18,608 copies/reaction; median approximately 332) and was detectable but below the limit of quantification (approximately 5 copies/reaction) in 1/62. Seventy-one other patients had 74 episodes with alternative diagnoses. Quantifiable HSP70 DNA was detectable in 6/74 (8%) episodes (range approximately 6-590 copies/reaction; median approximately 14) and detectable but below the limit of quantification in 34/74 (46%). Receiver-operator curve analysis (cut-off >10 copies/reaction) showed a clinical sensitivity of 98% (95% 91% to 100%) and specificity of 96% (95% CI 87% to 99%) for diagnosis of PCP. By contrast, clinical sensitivity of mt LSU rRNA PCR was 97% (95% CI 89% to 99%) and specificity was 68% (95% CI 56% to 78%). CONCLUSION: The HSP70 real-time PCR assay detects P. jirovecii DNA in BAL fluid and may have a diagnostic application. Quantification of P. jirovecii DNA by real-time PCR may also discriminate between colonisation with P. jirovecii and infection.

Real-time RT-PCR normalisation; strategies and considerations.

Real-time RT-PCR has become a common technique, no longer limited to specialist core facilities. It is in many cases the only method for measuring mRNA levels of vivo low copy number targets of interest for which alternative assays either do not exist or lack the required sensitivity. Benefits of this procedure over conventional methods for measuring RNA include its sensitivity, large dynamic range, the potential for high throughout as well as accurate quantification. To achieve this, however, appropriate normalisation strategies are required to control for experimental error introduced during the multistage process required to extract and process the RNA. There are many strategies that can be chosen; these include normalisation to sample size, total RNA and the popular practice of measuring an internal reference or housekeeping gene. However, these methods are frequently applied without appropriate validation. In this review we discuss the relative merits of different normalisation strategies and suggest a method of validation that will enable the measurement of biologically meaningful results.

The glutamate transporter GLAST-1 (EAAT-1) is expressed in the plasma membrane of osteocytes and is responsive to extracellular glutamate concentration.

The glutamate/aspartate transporter GLAST-1 is expressed in bone in vivo and also exists as a splice variant (GLAST-1a) in which exon 3 is excluded. Since GLAST-1 expression is regulated in bone in response to osteogenic mechanical stimuli in vivo and binding of glutamate to receptors on osteoblasts increases osteoblast number and activity in vitro, control of extracellular glutamate concentrations may be critical for balanced bone remodelling. To determine whether GLAST isoforms may act to regulate extracellular glutamate concentration in bone we investigated whether their pattern or level of expression is responsive to glutamate concentration in bone cells. GLAST-1a mRNA is expressed at lower levels than GLAST-1 mRNA in all cells examined. The GLAST-1a/GLAST-1 mRNA ratio is greater in MLO-Y4 osteocytes than in SaOS-2 osteoblast-like cells, although this does vary in SaOS-2 cells in response to extracellular glutamate concentration. Transfection of MLO-Y4 cells with green fluorescent protein (GFP)-tagged GLAST isoforms revealed a plasma membrane localization of GLAST-1, consistent with its transporter function, whereas GLAST-1a appeared to be expressed within internal vesicles. Interestingly, low extracellular glutamate concentrations redistributed GLAST-1-GFP into a similar internal expression pattern. Regulation of the expression and distribution of GLAST-1 by extracellular glutamate in bone cells indicates that it may regulate glutamate signalling in bone, consistent with its operation in the central nervous system.

Glutamate transporters in bone.

In the central nervous system Na(+)-dependent glutamate transporters bind extracellular glutamate and transport it into cells surrounding the synapse, terminating excitatory signals. These glutamate transporters also function as ion channels. The glutamate transporter, GLAST-1, is expressed in the plasma membrane of osteoblasts and osteocytes and is the same molecular weight as in brain. Thus in bone cells GLAST-1 may transport glutamate or operate as a glutamate gated ion channel. A splice variant, GLAST-1a, is also expressed in bone. Hydropathy and Western blot analysis suggest GLAST-1a adopts a reversed orientation within the cell membrane. Sodium and potassium ion gradients drive glutamate transport but glycosylation, oxidation and phosphorylation modulate transporter activity. Reversal of GLAST-1a would alter these modifications varying its transport activity under the same ionic gradients. The significance of GLAST-1/1a in bone in vivo is unknown. GLAST-1 knockout mice show no major disruption of skeletal development but have not been investigated in detail. Glutamate affects both osteoclast and osteoblast biology and the regulation of GLAST-1 by mechanical loading in bone suggests a role for glutamate transporters in osteogenesis. Differential regulation and modification of GLAST variants may provide an intricate mechanism controlling extracellular glutamate levels and thus its downstream signalling effects in bone.

The open reading frame of the Na(+)-dependent glutamate transporter GLAST-1 is expressed in bone and a splice variant of this molecule is expressed in bone and brain.

Previously we have reported expression of an mRNA with homology to the Na(+)-dependent glutamate transporter, GLAST-1, in bone. Here we demonstrate that the complete open reading frame of GLAST-1 mRNA and corresponding 69 kDa protein are expressed in rat bone in vivo. We have also discovered a novel splice variant (GLAST-1a), lacking exon 3, expressed in rat bone and brain. A 55 kDa protein detected by anti-GLAST antibody in rat cerebellum corresponds to the molecular weight of unglycosylated GLAST-1a. This has led us to propose that GLAST-1a has an opposite orientation in the cell membrane to GLAST-1.

CT findings of sternal fracture.

UNLABELLED: A retrospective study of blunt chest injuries due to motor vehicle accidents between the years 1990 and 1995 found 9 cases of sternal fracture that had both lateral plain film and thoracic axial CT scans performed. Plain film identified 8 of the 9 fractured sternums. CT scanning was only able to identify 6 sternal fractures. Secondary signs of sternal fracture were only seen in 5 of 9 patients, 3 had retrosternal haematomas and 2 had haematoma of the mediastinum. CONCLUSIONS: (1) plain film is still superior to Axial CT scanning for identification of sternal fracture; (2) retrosternal haematoma, although a specific sign for sternal fracture has low sensitivity (3/9); (3) mediastinal haematoma, a poorly specific sign for sternal fracture also demonstrated low sensitivity.