Plasmodium knowlesi has long been present in Malaysia, and is now an emerging cause of zoonotic human malaria. Cases have been confirmed throughout South-East Asia where the ranges of its natural macaque hosts and Anopheles leucosphyrus group vectors overlap. The majority of cases are from Eastern Malaysia, with increasing total public health notifications despite a concurrent reduction in Plasmodium falciparum and P. vivax malaria. The public health implications are concerning given P. knowlesi has the highest risk of severe and fatal disease of all Plasmodium spp in Malaysia. Current patterns of risk and disease vary based on vector type and competence, with individual exposure risks related to forest and forest-edge activities still poorly defined. Clustering of cases has not yet been systematically evaluated despite reports of peri-domestic transmission and known vector competence for human-to-human transmission.
Methods and analysis
A population-based case–control study will be conducted over a 2-year period at two adjacent districts in north-west Sabah, Malaysia. Confirmed malaria cases presenting to the district hospital sites meeting relevant inclusion criteria will be requested to enrol. Three community controls matched to the same village as the case will be selected randomly. Study procedures will include blood sampling and administration of household and individual questionnaires to evaluate potential exposure risks associated with acquisition of P. knowlesi malaria. Secondary outcomes will include differences in exposure variables between P. knowlesi and other Plasmodium spp, risk of severe P. knowlesi malaria, and evaluation of P. knowlesi case clustering. Primary analysis will be per protocol, with adjusted ORs for exposure risks between cases and controls calculated using conditional multiple logistic regression models.
This study has been approved by the human research ethics committees of Malaysia, the Menzies School of Health Research, Australia, and the London School of Hygiene and Tropical Medicine, UK.
Malaria due to Plasmodium knowlesi is reported throughout South-East Asia, and is the commonest cause of it in Malaysia. P. knowlesi replicates every 24 h and can cause severe disease and death. Current 2010 WHO Malaria Treatment Guidelines have no recommendations for the optimal treatment of non-severe knowlesi malaria. Artemisinin-combination therapies (ACT) and chloroquine have each been successfully used to treat knowlesi malaria; however, the rapidity of parasite clearance has not been prospectively compared. Malaysia's national policy for malaria pre-elimination involves mandatory hospital admission for confirmed malaria cases with discharge only after two negative blood films; use of a more rapidly acting antimalarial agent would have health cost benefits. P. knowlesi is commonly microscopically misreported as P. malariae, P. falciparum or P. vivax, with a high proportion of the latter two species being chloroquine-resistant in Malaysia. A unified ACT-treatment protocol would provide effective blood stage malaria treatment for all Plasmodium species.
Methods and analysis
ACT KNOW, the first randomised controlled trial ever performed in knowlesi malaria, is a two-arm open-label trial with enrolments over a 2-year period at three district sites in Sabah, powered to show a difference in proportion of patients negative for malaria by microscopy at 24 h between treatment arms (clinicaltrials.gov #NCT01708876). Enrolments started in December 2012, with completion expected by September 2014. A total sample size of 228 is required to give 90% power (α 0.05) to determine the primary end point using intention-to-treat analysis. Secondary end points include parasite clearance time, rates of recurrent infection/treatment failure to day 42, gametocyte carriage throughout follow-up and rates of anaemia at day 28, as determined by survival analysis.
Ethics and dissemination
This study has been approved by relevant institutional ethics committees in Malaysia and Australia. Results will be disseminated to inform knowlesi malaria treatment policy in this region through peer-reviewed publications and academic presentations.
Trial registration number
INFECTIOUS DISEASES; PARASITOLOGY
Simple and rapid extraction of human genomic DNA remains a bottle neck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab- and saliva samples. DNA is attracted on to a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle 4 microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for one month was demonstrated for captured DNA, facilitating straightforward collection, delivery and handling of genomic DNA in an environment-friendly protocol.
DNA extraction; Microtip; Electric field; Human genomic DNA; Human samples
The adaptor molecule SAP plays a critical role during natural killer T (NKT) cell development in humans and mice. In CD4+ T cells, SAP interacts with the tyrosine kinase Fyn to deliver signals required for TCR-induced TH2-type cytokine production. To determine whether the SAP-dependent signals controlling NKT cell ontogeny rely on its binding to Fyn, we used the OP9-DL1 system to initiate structure function studies of SAP in murine NKT cell development. In cultures containing wild type (WT) hematopoietic progenitors, we noted the transient emergence of cells that reacted with the NKT cell-specific agonist α-galactosyl ceramide (α-GC) and its analogue PBS57. Sap−/− cells failed to give rise to NKT cells in vitro; however, their development could be rescued by re-expression of WT SAP. Emergence of NKT cells was also restored by a mutant version of SAP (SAP R78A) that cannot bind to Fyn, but with less efficiency than WT SAP. This finding was accentuated in vivo in SapR78A knock-in mice as well as SapR78A competitive bone marrow chimeras, which retained NKT cells but at significantly reduced numbers compared to controls. Unlike SapR78A CD4+ T cells, which produce reduced levels of IL-4 following TCR ligation, α-GC-stimulated NKT cells from the livers and spleens of SapR78A mice produced TH2 cytokines and activated NK cells in a manner mimicking WT cells. Thus, SAP appears to use differential signaling mechanisms in NKT cells, with optimal ontogeny requiring Fyn binding, while functional responses occur independent of this interaction.
T cells; cell differentiation; signal transduction
Quality of life (QL) is an important consideration when comparing adjuvant therapies for early breast cancer, especially if they differ substantially in toxicity. We evaluated QL and Q-TWiST among patients randomised to adjuvant dose-intensive epirubicin and cyclophosphamide administered with filgrastim and progenitor cell support (DI-EC) or standard-dose anthracycline-based chemotherapy (SD-CT). We estimated the duration of chemotherapy toxicity (TOX), time without disease symptoms and toxicity (TWiST), and time following relapse (REL). Patients scored QL indicators. Mean durations for the three transition times were weighted with patient reported utilities to obtain mean Q-TWiST. Patients receiving DI-EC reported worse QL during TOX, especially treatment burden (month 3: P<0.01), but a faster recovery 3 months following chemotherapy than patients receiving SD-CT, for example, less coping effort (P<0.01). Average Q-TWiST was 1.8 months longer for patients receiving DI-EC (95% CI, −2.5 to 6.1). Q-TWiST favoured DI-EC for most values of utilities attached to TOX and REL. Despite greater initial toxicity, quality-adjusted survival was similar or better with dose-intensive treatment as compared to standard treatment. Thus, QL considerations should not be prohibitive if future intensive therapies show superior efficacy.
breast cancer; adjuvant chemotherapy; quality of life; adaptation; quality-adjusted survival
To better understand the pathogenesis of human herpesvirus 6 (HHV-6), it is important to elucidate the functional aspects of immediate-early (IE) genes at the initial phase of the infection. To study the functional role of the HHV-6B IE gene encoding U95, we generated a U95-Myc fusion protein and screened a pretransformed bone marrow cDNA library for U95-interacting proteins, using yeast-two hybrid analysis. The most frequently appearing U95-interacting protein identified was GRIM-19, which belongs to the family of genes associated with retinoid-interferon mortality and serves as an essential component of the oxidative phosphorylation system. This interaction was verified by both coimmunoprecipitation and confocal microscopic coimmunolocalization. Short-term HHV-6B infection of MT-4 T-lymphocytic cells induced syncytial formation, resulted in decreased mitochondrial membrane potential, and led to progressively pronounced ultrastructural changes, such as mitochondrial swelling, myelin-like figures, and a loss of cristae. Compared to controls, RNA interference against U95 effectively reduced the U95 mRNA copy number and abrogated the loss of mitochondrial membrane potential. Our results indicate that the high affinity between U95 early viral protein and GRIM-19 may be closely linked to the detrimental effect of HHV-6B infection on mitochondria. These findings may explain the alternative cell death mechanism of expiration, as opposed to apoptosis, observed in certain productively HHV-6B-infected cells. The interaction between U95 and GRIM-19 is thus functionally and metabolically significant in HHV-6B-infected cells and may be a means through which HHV-6B modulates cell death signals by interferon and retinoic acid.
Objective: To examine the impact for the UK population of providing statin treatment for diabetic patients for the primary prevention of coronary heart disease at a coronary event risk lower than currently recommended by the National Service Framework (NSF) for coronary heart disease.
Design: Cross sectional survey.
Setting: England 1998.
Participants: Nationally representative sample of 6879 subjects aged 35–74 years living in private households.
Main outcome measures: The proportion of the UK population recommended for statin treatment according to the NSF for coronary heart disease, and the proportion of the population with diabetes at a coronary disease event risk of ≥ 15% over 10 years.
Results: Of the 6879 subjects with total cholesterol measurements, 218 (3.2%) had diabetes mellitus. In this nationally representative sample, 6.3% of the subjects (95% confidence interval (CI), 5.7% to 6.9%) were candidates for statin treatment for the secondary prevention of coronary heart disease, including 0.7% (95% CI 0.5% to 0.9%) with diabetes. A further 2.4% (95% CI 2.0% to 2.8%), including 0.4% (0.2% to 0.6%) with diabetes, were identified as candidates for primary prevention of coronary heart disease according to the NSF for coronary heart disease. Lowering the primary prevention threshold for statin treatment to a coronary event risk of ≥ 15% over 10 years in diabetic patients identified an additional 0.5% of the population.
Conclusions: Extending statin treatment to diabetic patients at a coronary heart disease risk of ≥ 15% over 10 years would have a relatively small numerical impact in the UK population. Thus patients with diabetes mellitus should, as a minimum, be targeted for statin treatment at this level of risk.
diabetes; coronary heart disease; risk estimate; statins
hepatitis B virus reactivation; chemotherapy; risk factors
Understanding of cellular processes and underlying molecular events requires knowledge about different aspects of molecular interactions, networks of molecules and pathways in addition to the sequence, structure and function of individual molecules involved. Databases of interacting molecules, pathways and related chemical reaction equations have been developed. The kinetic data for these interactions, which is important for mechanistic investigation, quantitative study and simulation of cellular processes and events, is not provided in the existing databases. We introduce a new database of Kinetic Data of Bio-molecular Interactions (KDBI) aimed at providing experimentally determined kinetic data of protein–protein, protein-RNA, protein-DNA, protein-ligand, RNA-ligand, DNA-ligand binding or reaction events described in the literature. KDBI contains information about binding or reaction event, participating molecules (name, synonyms, molecular formula, classification, SWISS-PROT AC or CAS number), binding or reaction equation, kinetic data and related references. The kinetic data is in terms of one or a combination of the following quantities as given in the literature of a particular event: association/dissociation or on/off rate constant, first/second/third/… order rate constant, equilibrium rate constant, catalytic rate constant, equilibrium association/dissociation constant, inhibition constant and binding affinity constant. Each entry can be retrieved through protein or nucleic acid or ligand name, SWISS-PROT AC number, ligand CAS number and full-text search of a binding or reaction event. KDBI currently contains 8273 entries of biomolecular binding or reaction events involving 1380 proteins, 143 nucleic acids and 1395 small molecules. Hyperlinks are provided for accessing references in Medline and available 3D structures in PDB and NDB. This database can be accessed at http://xin.cz3.nus.edu.sg/group/kdbi/kdbi.asp.
OBJECTIVES—To estimate the cost effectiveness of statin treatment in preventing coronary heart disease (CHD) and to examine the effect of the CHD risk level targeted and the cost of statins on the cost effectiveness of treatment.
DESIGN—Cohort life table method using data from outcome trials.
MAIN OUTCOME MEASURES—The cost per life year gained for lifelong statin treatment at annual CHD event risks of 4.5% (secondary prevention) and 3.0%, 2.0%, and 1.5% (all primary prevention), with the cost of statins varied from £100 to £800 per year.
RESULTS—The costs per life year gained according to annual CHD event risk were: for 4.5%, £5100; 3.0%, £8200; 2.0%, £10 700; and 1.5%, £12 500. Reducing the cost of statins increases cost effectiveness, and narrows the difference in cost effectiveness across the range of CHD event risks.
CONCLUSIONS—At current prices statin treatment for secondary prevention, and for primary prevention at a CHD event risk 3.0% per year, is as cost effective as many treatments in wide use. Primary prevention at lower CHD event risks (< 3.0% per year) is less cost effective and unlikely to be affordable at current prices and levels of health service funding. As the cost of statins falls, primary prevention at lower risk levels becomes more cost effective. However, the large volume of treatment needed will remain a major problem.
Keywords: coronary artery disease; cost effectiveness; statins; primary prevention; secondary prevention
Objective—To examine the validity of estimates of coronary heart disease (CHD) risk by the Framingham risk function, for European populations.
Design—Comparison of CHD risk estimates for individuals derived from the Framingham, prospective cardiovascular Münster (PROCAM), Dundee, and British regional heart (BRHS) risk functions.
Setting—Sheffield Hypertension Clinic.
Patients—206 consecutive hypertensive men aged 35-75 years without preexisting vascular disease.
Results—There was close agreement among the Framingham, PROCAM, and Dundee risk functions for average CHD risk. For individuals the best correlation was between Framingham and PROCAM, both of which use high density lipoprotein (HDL) cholesterol. When Framingham was used to target a CHD event rate > 3% per year, it identified men with mean CHD risk by PROCAM of 4.6% per year and all had CHD event risks > 1.5% per year. Men at lower risk by Framingham had a mean CHD risk by PROCAM of 1.5% per year, with 16% having a CHD event risk > 3.0% per year. BRHS risk function estimates of CHD risk were fourfold lower than those for the other three risk functions, but with moderate correlations, suggesting an important systematic error.
Conclusion—There is close agreement between the Framingham, PROCAM, and Dundee risk functions as regards average CHD risk, and moderate agreement for estimates within individuals. Taking PROCAM as the external standard, the Framingham function separates high and low CHD risk groups and is acceptably accurate for northern European populations, at least in men.
Keywords: ischaemic heart disease; prevention; risk factors
A 67 year old woman was admitted with a three week history of vomiting, having become increasingly confused for three days. Investigations revealed deranged serum biochemistry consistent with a combination of a diabetic non-ketotic hyperosmolar state and a metabolic alkalosis consistent with gastric outflow obstruction. She was treated with intravenous saline, intravenous insulin, and subcutaneous heparin, but did not improve clinically and had an asystolic cardiac arrest the following day; she was transferred to the intensive care unit and despite treatment with inotropes she died 40 hours after admission. Necropsy revealed that the stomach was massively dilated with gas and stomach contents, and contained many small black faceted gall stones. In addition a large nonfaceted brown-yellow gall stone was wedged in the pyloric antrum causing total obstruction. The patient had died from a complex metabolic derangement including non-ketotic hyperosmotic diabetic coma and metabolic alkalosis precipitated by the acute gastric outflow obstruction complicated by previously undiagnosed type II diabetes mellitus.
An unusual case of small cell variant of Ki-1 non-Hodgkin's lymphoma diagnosed one year after an original diagnosis of idiopathic myelofibrosis is reported. On the second occasion, the patient presented with fever, lymphadenopathy and hepatosplenomegaly. A lymph node biopsy specimen confirmed a diagnosis of small cell variant of Ki-1 lymphoma. A repeat bone marrow biopsy specimen showed myelofibrosis with no evidence of lymphomatous infiltration, but cytogenetic studies on blood, bone marrow and skin fibroblasts revealed a novel chromosomal translocation t(3,4)(q13;q12).
A 43 year old man with inoperable aortic coarctation and severe hypertension requiring near maximal anti-hypertensive treatment was admitted in severe heart failure. After 2 weeks of treatment the heart failure and blood pressure were incompletely controlled and angiotensin converting enzyme (ACE) inhibitor was started. Serum creatinine was normal before starting the ACE inhibitor and on discharge from hospital. The patient was re-admitted a week later with gross fluid retention and in renal failure. In the absence of alternative causes, a diagnosis of ACE inhibitor-induced renal failure was made and treatment was stopped. The patient required haemodialysis for 2 days and within 1 week the renal function had reverted to normal and has remained so for 1 year. We propose that the renal haemodynamics in severe aortic coarctation are similar to those in bilateral severe renal artery stenosis and advise caution in the use of ACE inhibitors for adults with aortic coarctation.
Serum levels of alphafetoprotein are raised in 60-80% of patients with hepatocellular carcinoma. Although widely used as a serum marker, frequent false-positive results in patients with benign liver disease, result in poor specificity. This occurs particularly when levels of alphafetoprotein fall between 50-500 ng ml-1, the so-called 'grey area'. Recent reports suggest that isoelectric focusing of alphafetoprotein demonstrates certain bands that are more specific for hepatocellular carcinoma. Our aim was to determine whether the apparent specificity of this new approach is gained at the expense of decreased sensitivity. Sera from 110 patients with a 'non-diagnostic' serum alphafetoprotein level (50-500 ng ml-1) were examined by isoelectric focusing and quantified by densitometric scanning. Ten patients with chronic liver disease and a raised serum alphafetoprotein level (50-500 ng ml-1), but with no evidence of hepatocellular carcinoma, were also studied. Isoelectric focusing revealed characteristic hepatocellular carcinoma bands (bands +II and +III) in 96% patients overall, and 100% of those with levels of total alphafetoprotein greater than 100 ng ml-1. No such bands were seen among ten subjects with cirrhosis but without hepatocellular carcinoma. Bands that are characteristic of hepatocellular carcinoma (bands +II or +III) are seen in the great majority of hepatocellular carcinoma patients; their absence makes a diagnosis of hepatocellular carcinoma extremely unlikely.
The association between haemospermia and hypertension was examined in a case-control study comparing 5 hypertensive patients with haemospermia to 20 age-matched hypertensive men. Patients with haemospermia had much higher blood pressures than hypertensive controls (200/131 mmHg vs 147/90 mmHg; P less than 0.0005/P less than 0.0001), higher left ventricular voltage on ECG (P less than 0.02), and higher concentrations of serum creatinine, proteinuria and renovascular disease (all P = 0.06 vs controls). Haemospermia is associated with severe uncontrolled hypertension. It is not, however, associated with hypertension per se, as the prevalence of hypertension in published series of patients with haemospermia is no higher than that expected in the general population. Men presenting with haemospermia should have their blood pressure measured carefully as they may require antihypertensive treatment urgently.
The purpose of this study was to identify whether a past history of childhood sexual abuse is a risk factor for repeated Deliberate Self-Harm (DSH). The study was a 6-month prospective study of 178 patients responsible for 190 consecutive cases of DSH seen during a 3-month censoring period. Patients were identified by review of the in-patient and accident and emergency (A&E) records of all cases of DSH at the A&E department of a major teaching hospital. Patients with a history of childhood sexual abuse showed a marked clustering of four major risk factors for repeat DSH (unemployment, past deliberate self-poisoning, self-injury and psychiatric illness) and were significantly more likely to repeat DSH within the 6-month follow-up period.