Mutation detection rate and spectrum in familial hypercholesterolaemia patients in the UK pilot cascade project.

Cascade testing using DNA-mutation information is now recommended in the UK for patients with familial hypercholesterolaemia (FH). We compared the detection rate and mutation spectrum in FH patients with a clinical diagnosis of definite (DFH) and possible (PFH) FH. Six hundred and thirty-five probands from six UK centres were tested for 18 low-density lipoprotein receptor gene (LDLR) mutations, APOB p.Arg3527Gln and PCSK9 p.Asp374Tyr using a commercial amplification refractory mutation system (ARMS) kit. Samples with no mutation detected were screened in all exons by single strand conformation polymorphism analysis (SSCP)/denaturing high performance liquid chromatography electrophoresis (dHPLC)/direct-sequencing, followed by multiplex ligation-dependent probe amplification (MLPA) to detect deletions and duplications in LDLR.The detection rate was significantly higher in the 190 DFH patients compared to the 394 PFH patients (56.3% and 28.4%, p > 0.00001). Fifty-one patients had inadequate information to determine PFH/DFH status, and in this group the detection rate was similar to the PFH group (25.5%, p = 0.63 vs PFH). Overall, 232 patients had detected mutations (107 different; 6.9% not previously reported). The ARMS kit detected 100 (44%) and the MLPA kit 11 (4.7%). Twenty-eight (12%) of the patients had the APOB p.Arg3527Gln and four (1.7%) had the PCSK9 p.Asp374Tyr mutation. Of the 296 relatives tested from 100 families, a mutation was identified in 56.1%. In 31 patients of Indian/Asian origin 10 mutations (two previously unreported) were identified. The utility of the ARMS kit was confirmed, but sequencing is still required in a comprehensive diagnostic service for FH. Even in subjects with a low clinical suspicion of FH, and in those of Indian origin, mutation testing has an acceptable detection rate.

Family tracing to identify patients with familial hypercholesterolaemia: the second audit of the Department of Health Familial Hypercholesterolaemia Cascade Testing Project.

BACKGROUND: Family tracing is a method recognized to find new patients with familial hypercholesterolaemia (FH). We have implemented family tracing led by FH Nurses and have determined acceptability to patients, feasibility and costs. METHODS: Nurses were located at five National Health Service (NHS) Trusts; they identified FH patients and offered them family tracing. Responses and test results were recorded on a database and summarized on a family pedigree. RESULTS: The majority ( approximately 70%) of index cases participated; the proportion was lower when patients had been discharged from the clinics and in metropolitan areas. On average, 34% (range 13-50%) of relatives lived outside the catchment area of the clinics and could not attend the nurse-led FH clinics. Of the previously untested relatives, 76% who lived in the catchment area of the clinic came forward to be tested. One-third of the relatives who came forward for testing were children

Are patients with familial hypercholesterolaemia well managed in lipid clinics? An audit of eleven clinics from the Department of Health Familial Hypercholesterolaemia Cascade Testing project.

BACKGROUND: Familial hypercholesterolaemia (FH) is an autosomal co-dominant disorder which is relatively common, leads to high levels of LDL-cholesterol and if untreated to early coronary heart disease. An audit of current practice at National Health Service Trusts in England was undertaken to determine whether FH patients meet the diagnostic criteria for FH; are being offered appropriate advice and treatment; and to what extent their families are contacted and offered testing for the disorder. METHODS: Medical records of known FH patients (over 18 years of age and diagnosed before 31 December 2003) were accessed to obtain information on diagnosis, treatment and family tracing. RESULTS: The records of 733 FH patients were examined, 79% met the UK 'Simon Broome' register criteria for the diagnosis of definite or possible FH. Analyses showed that patients were usually offered appropriate advice and treatment, with 89% being on a statin. However, the audit indicated a high variability in family tracing between the sites, with significant differences in the frequency of inclusion of a family pedigree in the notes (range 1-71%, mean 35%); the general practitioner (GP) being advised that first-degree relatives should be tested (range 4-52%, mean 27%); and the proportion of relatives contacted and tested (range 6-50%, mean 32%). CONCLUSION: FH patients are well cared for in lipid clinics in England, are being given appropriate lifestyle advice and medication, but an increase in recording of LDL-cholesterol levels may lead to improvements in their management. Practice in family tracing appears to vary widely between clinics.

A novel coloured latex test for the detection and identification of more than one antigen.

A novel latex agglutination test employing coloured latex particles is described. The latex reagent contains two or more differently coloured latex suspensions, each colour having been coated before mixing with at least one specific antibody, so that two or more antigens can be detected and identified with a single reagent. The antigen is identified by observing the colour of the agglutinated particles against a contrasting change in the colour of the background.

Enzyme-linked immunosorbent assay (ELISA) to detect antibodies in gonorrhea using whole cells.

The choice of an antigen that will adequately differentiate between infected and non-infected patients has been a problem in detecting gonococcal antibodies for diagnosis. We have used the sensitive technique of ELISA to test various serotypes of Neisseria gonorrhoeae for their suitability as antigens. Whole cells of each serotype were attached to polystyrene plates using poly-L-lysine, N gonorrhoeae, strain H1 type 1 was used to detect antibodies in patients with known clinical history and then as a standard to evaluate the ability of different serotypes to differentiate between infected and non-infected groups.

The effects of age on patient-reported outcome measures in total knee replacements.

We present a comparison of patient-reported outcomes (PROMs) in relation to patient age, in patients who had received a total (TKR) or unicompartmental knee replacement (UKR). The outcome was evaluated using the Oxford knee score (OKS), EuroQol (EQ-5D) and satisfaction scores. Patients aged 65 to 84 years demonstrated better pre-operative function scores than those aged < 65 years (OKS, p = 0.03; EQ-5D, p = 0.048) and those aged ≥ 85 years (OKS, p = 0.03). Post-operative scores were comparable across age groups, but a linear trend for greater post-operative improvement in OKS and EQ-5D was seen with decreasing age (p < 0.033). The overall mean satisfaction score at six months was 84.9, but those aged < 55 years exhibited a lower mean level of satisfaction (78.3) compared with all other age groups (all p < 0.031). The cumulative overall two-year revision rate was 1.3%. This study demonstrates that good early outcomes, as measured by the OKS and EQ-5D, can be anticipated following knee replacement regardless of the patient's age, although younger patients gain greater improvement. However, the lower satisfaction in those aged < 55 years is a concern, and suggests that outcome is not fully encapsulated by the OKS and EQ-5D evaluation, and raises the question whether the OKS alone is an appropriate measure of pain and function in younger, more active individuals.

Long-term trends in the Oxford knee score following total knee replacement.

The Oxford knee score (OKS) is a validated and widely accepted disease-specific patient-reported outcome measure, but there is limited evidence regarding any long-term trends in the score. We reviewed 5600 individual OKS questionnaires (1547 patients) from a prospectively-collected knee replacement database, to determine the trends in OKS over a ten-year period following total knee replacement. The mean OKS pre-operatively was 19.5 (95% confidence interval (CI) 18.8 to 20.2). The maximum post-operative OKS was observed at two years (mean score 34.4 (95% CI 33.7 to 35.2)), following which a gradual but significant decline was observed through to the ten-year assessment (mean score 30.1 (95% CI 29.1 to 31.1)) (p < 0.001). A similar trend was observed for most of the individual OKS components (p < 0.001). Kneeling ability initially improved in the first year but was then followed by rapid deterioration (p < 0.001). Pain severity exhibited the greatest improvement, although residual pain was reported in over two-thirds of patients post-operatively, and peak improvement in the night pain component did not occur until year four. Post-operative OKS was lower for women (p < 0.001), those aged < 60 years (p < 0.003) and those with a body mass index > 35 kg/m(2) (p < 0.014), although similar changes in scores were observed. This information may assist surgeons in advising patients of their expected outcomes, as well as providing a comparative benchmark for evaluating longer-term outcomes following knee replacement.

Patient-reported complications after elective joint replacement surgery: are they correct?

Using general practitioner records and hospital notes and through direct telephone conversation with patients, we investigated the accuracy of nine patient-reported complications gathered from a self-completed questionnaire after elective joint replacement surgery of the hip and knee. A total of 402 post-discharge complications were reported after 8546 elective operations that were undertaken within a three-year period. These were reported by 136 men and 240 women with a mean age of 71.8 years (34 to 93). A total of 319 reported complications (79.4%; 95% confidence interval 75.4 to 83.3) were confirmed to be correct. High rates of correct reporting were demonstrated for infection (94.5%) and the need for further surgery (100%), whereas the rates of reporting deep-vein thrombosis (DVT), pulmonary embolism, myocardial infarction and stroke were lower (75% to 84.2%). Dislocation, peri-prosthetic fractures and nerve palsy had modest rates of correct reporting (36% to 57.1%). More patients who had knee surgery delivered incorrect reports of dislocation (p = 0.001) and DVT (p = 0.013). Despite these variations, it appears that post-operative complications may form part of a larger patient-reported outcome programme after elective joint replacement surgery.

Analysis of antibodies in local and disseminated Neisseria gonorrhoeae infections by means of gel electrophoresis-derived ELISA.

Major antigens in Neisseria gonorrhoeae were identified by surface labelling the organisms with 125I and electrophoresing extracts in polyacrylamide with sodium dodecyl sulphate. Horizontal slices of the gels were cut out and tested in individual wells against patients' sera using ELISA. Serum from local gonococcal infections reacted with Protein II and, probably, lipopolysaccharide, but not with Protein I in deoxycholate (DOC) extracts and gave no reaction with Triton X-100 extracts. Serum from disseminated gonococcal infections reacted with Protein I in the DOC extract and with pili and a number of undefined possibly cytoplasmic membrane antigens in the Triton X-100 extract. The significance of the results and the potential of the method are discussed.

Application of immuno-blotting to the differentiation of specific antibodies in gonorrhoea.

The technique of immuno-blotting was used to examine the specificity of serum antibodies in patients with gonorrhoea and to define the significant antigenic determinant present in the desoxycholate extracts of gonococcal cell walls used previously for serological diagnosis. IgG but not IgA antibody was directed predominantly against lipopolysaccharide. Antibodies to outer membrane proteins were not found as frequently as expected. There was a suggestion from the results that antibodies to antigen PI was more common in systemic than in local infections.

Effects of trialkyllead compounds on growth, respiration and ion transport in Escherichia coli K12.

Triethyllead and tripropyllead cations affected growth, energy metabolism and ion transport in Escherichia coli K12. The tripropyllead compound was more liposoluble than the triethyl analogue and was also more effective in inhibiting cell growth and the oxygen uptake of both intact cells and membrane particles. Triethyllead acetate (5 microM) inhibited growth on non-fermentable carbon sources, such as glycerol and succinate, more markedly than on glucose. At higher concentrations, triethyllead caused significant inhibition of respiration rates of intact cells; the concentration giving 50% inhibition was 60 microM for glycerol-grown cells and 150 microM for glucose-grown cells. Oxidation of succinate by membrane particles was less sensitive to inhibition by the tripropyl- or triethyllead compounds than were the oxidations of DL-lactate or NADH. Triethyllead acetate [1.9 mumol (mg membrane protein)-1] inhibited the reduction by NADH of cytochromes; evidence for more than one site of inhibition in the respiratory chain was obtained. Membrane-bound ATPase activity was strongly inhibited by triethyllead acetate in the absence or presence of Cl-. The concentration of inhibitor giving 50% inhibition [0.02 mumol (mg membrane protein)-1] was about two orders of magnitude lower than that required for 50% inhibition of substrate oxidation rates in membranes. Triethyllead acetate (1 microM) induced swelling of spheroplasts in iso-osmotic solutions of either NH4Cl or NH4Br, presumably as a result of the mediation by the organolead compound of Cl-/OH- and Br-/OH- antiports across the cytoplasmic membrane. Similar exchanges of OH- for F-, NO3- or SO4(2)- or the uniport of H+ could not be demonstrated. Comparisons are drawn between the effects of trialkyllead compounds and those of the more widely studied trialkyltin compounds.

Direct detection of groups A, C and G streptococci in clinical specimens by a trivalent colour test.

A simple trivalent colour test, developed for the rapid detection and identification of streptococci belonging to Lancefield groups A, C and G, was evaluated for sensitivity and specificity with cultures and when directly used with wound and throat swabs. In tests performed on cultures, all of 94 group A, 78 group C and 94 group G cultures were correctly identified. In direct tests on wound swabs, 49 of 52 group A, 17 of 19 group C and 48 of 51 group G streptococci were detected and correctly identified; no false positives were observed. With throat swabs from pharyngitis patients 34 of 36 group A, 3 of 6 group C and 5 of 8 group G streptococci gave positive results. Almost 10% of these swabs gave false positive reactions with the group C component of the test system. Samples taken from uninfected individuals indicated that the false positives were probably associated with blood group A. The test system gives rapid and reliable results with streptococcal cultures, but when directly applied to clinical samples the results must be interpreted with caution, particularly if the patient's blood group is not known.

Novel color test for rapid detection of group A streptococci.

A novel color test for the rapid detection of group A streptococci has been developed. The test, designed to be suitable for use in clinical laboratories as well as by less experienced personnel, incorporates the simplicity of latex tests with a color change to indicate the presence of group A streptococcal antigen. The test, which takes 5 min, was evaluated with 646 throat swabs, with a 15.6% incidence of group A streptococci; for swabs which yielded 10 or more group A streptococcal colonies in cultures, the sensitivity was 96.8%, and the specificity was 99.1%. In addition, the color test was 100% sensitive and specific when used to detect group A streptococcal antigen in beta-hemolytic colonies from culture plates.

The specificity of serum and local antibodies in female gonorrhoea.

Immunoblotting has been used to compare the specificity of serum and local IgG and IgA antibodies in 13 women with gonorrhoea and in 13 controls. The technique allowed the simultaneous detection of antibodies to the major outer membrane proteins I, II, and III, pili and lipopolysaccharide; antibodies to another antigen which is probably a 'carbohydrate' were also detected. Serum and local IgG and IgA were found to be produced to several antigens during gonococcal infections, although the quantity of antibody was greater in serum. There was little change in the specificity of serum antibodies whereas the local response to LPS and pili increased over the two week study period. Serum antibody to LPS was more often IgG than IgA. Sera contained antibodies to 'carbohydrate', pili and lipopolysaccharide (LPS) whilst the local response was largely to the latter two antigens. Antibody to the outer membrane proteins was rarely detected. Control sera, but not vaginal washings, contained IgG and IgA to the major antigens but the staining of the immunoblots was less intense than those from patient's sera suggesting quantitative differences.