Fluorogenic substrate detection of viable intracellular and extracellular pathogenic protozoa.

Viable Leishmania promastigotes and amastigotes were detected by epifluorescence microscopy with fluorescein diacetate being used to mark living parasites and the nucleic acid-binding compound ethidium bromide to stain dead cells. This procedure is superior to other assays because it is faster and detects viable intracellular as well as extracellular Leishmania. Furthermore, destruction of intracellular pathogens by macrophages is more accurately determined with fluorescein diacetate than with other stains. The procedure may have applications in programs to develop drugs and vaccines against protozoa responsible for human and animal disease.

A novel size-selective airborne particle size fractionating instrument for health risk evaluation.

Health risks associated with the inhalation of airborne particles are known to be influenced by particle size. Studies have shown that certain nanoparticles, with diameters <100 nm, have increased toxicity relative to larger particles of the same substance. A reliable, size-resolving sampler able to collect a wide range of particle sizes, including particles with sizes in the nanometre range, would be beneficial in investigating health risks associated with the inhalation of airborne particles. A review of current aerosol samplers used for size-resolved collection of airborne particles highlighted a number of limitations. These could be overcome by combining an inertial deposition impactor with a diffusion collector in a single device. Verified theories of diffusion and inertial deposition suggested an optimal design and operational regime. The instrument was designed for analysing mass distribution functions. Calibration was carried out using a number of recognized techniques. The sampler was tested in the field by collecting size-resolved samples of lead containing aerosols present at workplaces in factories producing crystal glass. The mass deposited on each screen proved sufficient to be detected and measured by an appropriate analytical technique. Mass concentration distribution functions of lead were produced. The nanofraction of lead in air varied from 10 to 70% by weight of total lead.

Accurate Patient-Specific Machine Learning Models of Glioblastoma Invasion Using Transfer Learning.

BACKGROUND AND PURPOSE: MR imaging-based modeling of tumor cell density can substantially improve targeted treatment of glioblastoma. Unfortunately, interpatient variability limits the predictive ability of many modeling approaches. We present a transfer learning method that generates individualized patient models, grounded in the wealth of population data, while also detecting and adjusting for interpatient variabilities based on each patient's own histologic data. MATERIALS AND METHODS: We recruited patients with primary glioblastoma undergoing image-guided biopsies and preoperative imaging, including contrast-enhanced MR imaging, dynamic susceptibility contrast MR imaging, and diffusion tensor imaging. We calculated relative cerebral blood volume from DSC-MR imaging and mean diffusivity and fractional anisotropy from DTI. Following image coregistration, we assessed tumor cell density for each biopsy and identified corresponding localized MR imaging measurements. We then explored a range of univariate and multivariate predictive models of tumor cell density based on MR imaging measurements in a generalized one-model-fits-all approach. We then implemented both univariate and multivariate individualized transfer learning predictive models, which harness the available population-level data but allow individual variability in their predictions. Finally, we compared Pearson correlation coefficients and mean absolute error between the individualized transfer learning and generalized one-model-fits-all models. RESULTS: Tumor cell density significantly correlated with relative CBV (r = 0.33, P < .001), and T1-weighted postcontrast (r = 0.36, P < .001) on univariate analysis after correcting for multiple comparisons. With single-variable modeling (using relative CBV), transfer learning increased predictive performance (r = 0.53, mean absolute error = 15.19%) compared with one-model-fits-all (r = 0.27, mean absolute error = 17.79%). With multivariate modeling, transfer learning further improved performance (r = 0.88, mean absolute error = 5.66%) compared with one-model-fits-all (r = 0.39, mean absolute error = 16.55%). CONCLUSIONS: Transfer learning significantly improves predictive modeling performance for quantifying tumor cell density in glioblastoma.

Caffeine urinary metabolite ratios as markers of enzyme activity: a theoretical assessment.

At least six urinary metabolite ratios of caffeine have been proposed as probes for in vivo CYP1A2 activity and three for in vivo NAT2 activity. Claims for the frequency distribution of the activity of CYP1A2 based on these empirical ratios have varied from log-normal to trimodal. We have examined the validity of these nine ratios by developing computer simulations using values reported in the literature for the kinetic parameters of caffeine and its metabolites. The results show that the sensitivity of the ratios to confounding variables is, in some cases, greater than their sensitivity to the activity of the enzyme that they are intended to mark. The six CYP1A2 ratios did not exhibit the same pattern of dependency on confounding variables which, in turn, resulted in different shapes of population distributions for each ratio as enzyme activity was varied systematically. Although the dependency of the three NAT2 ratios on confounding variable was less marked, they also showed different patterns of dependency. The outcomes of the simulations were consistent with much of the experimental data on caffeine metabolite ratios. To support the findings from the simulations, simplified equations for each metabolite ratio were derived which emphasize the dominant determinants. With some of the CYP1A2 ratios urine flow was significant to the point where its variance and heterogeneity between populations could lead to spurious detection of polymorphism in CYP1A2 function. Also, if the variability of a dominant confounding factor was high and sensitivity of the ratio to intrinsic CYP1A2 activity was low, any polymorphism in the latter would be obscured. When a specific time interval was defined for urine collection, this time was shown to be a critical factor in the ability to discriminate bimodality in some of the ratios, when a marked polymorphism in enzyme activity was assumed. Those ratios which have shown no evidence for bimodality in CYP1A2 function in experimental studies are inherently more discriminant of such heterogeneity compared to those ratios which have been claimed to detect polymorphism of CYP1A2 from experimental data. While recommending a 'best buy' from amongst the caffeine urinary metabolite ratios, we favour plasma/saliva indices (caffeine half-life or paraxanthine/caffeine ratio in a spot sample).

Evidence for a dissociation in the control of sparteine, debrisoquine and metoprolol metabolism in Nigerians.

The 0-8 hour urinary distributions of the metabolic ratios of sparteine (100 mg), debrisoquine (10 mg) and metoprolol (100 mg) were measured in 165 healthy, unrelated, black Nigerian medical students. There was a weak correlation (rs = 0.51, p < 0.001; n = 82) between the metoprolol/alpha-hydroxymetoprolol (M/HM) and the sparteine/total (2- + 5-) dehydrosparteine (S/DHS) ratios. No significant correlations were found between the debrisoquine/4-hydroxydebrisoquine (D/HD) and M/HM ratios (rs = 0.16, n = 33) and between the D/HD and S/DHS ratios (rs = 0.31, n = 38). Both visual inspection and kernel density analysis of the data suggested the presence of two phenotypic groups for sparteine oxidation, with 4% of the population studied being putative poor metabolizers. In contrast biomodality was not apparent in the distribution of the log10M/HM and log10D/HD ratios. These findings provide evidence for a dissociation in the control of metoprolol, sparteine and debrisoquine oxidation in Nigerians and highlight the difficulties in the interpretation of data from pharmacogenetic studies in different ethnic groups.

Altered plasma drug binding in cancer: role of alpha 1-acid glycoprotein and albumin.

Altered concentrations of serum proteins often accompany malignant disease. The effect of these changes on drug binding was studied with lidocaine, a basic drug, and tolbutamide, an acidic drug. Patients with cancer had increased serum concentrations of the acute-phase protein alpha 1-acid glycoprotein (AAG) and lowered serum concentration of albumin. In association with these changes lidocaine binding was increased at all concentrations studied (predialysis concentrations 2, 6, and 10 microgram . ml-1) and that of tolbutamide was decreased at the highest concentration (200 microgram . ml-1). Not all of the increase in lidocaine binding was explicable on the basis of increased serum AAG concentration. Estimation of binding parameters with a model with two independent sites showed increased affinity at the high affinity site in cancer patients with no change in the calculated number of binding sites. Therefore, in cancer there is increased lidocaine binding in association with increased AAG concentrations. We also record the novel observation of a change in the intrinsic properties of the high affinity binding site.

Protein binding of aspirin and salicylate measured by in vivo ultrafiltration.

OBJECTIVE: Methods for measuring protein binding of drugs generally require direct measurement of the concentration of unbound drug and thus may require a highly sensitive assay. In vivo ultrafiltration has been used to determine protein binding of endogenous substances. We have examined its value for measuring protein binding of drugs because it requires measurement of only the concentration of total drug, not unbound drug, in plasma. METHODS: The protein binding of aspirin and its metabolite salicylate was measured in 29 healthy subjects 20 minutes after a single oral dose of 600 mg soluble aspirin, by the new method, in vivo ultrafiltration, as well as by a standard method, in vitro ultracentrifugation. RESULTS: The data for salicylate were examined systematically to determine the optimal method of determining estimates of protein binding by in vivo ultrafiltration. Estimates of protein binding of salicylate were 81.7% +/- 10.1% (mean +/- SD) by the in vivo method and 81.6% +/- 11.3% by in vitro ultracentrifugation. Bland-Altman analysis of agreement showed that within-individual differences in percentage of protein binding determined by the two methods did not differ significantly from zero (mean difference, 0.07%; 95% confidence interval, -2.33 to +2.46). There was a highly significant correlation between estimates of protein binding by the two methods (r = 0.82; p = 0.001). Protein binding of aspirin was estimated of protein binding by the two methods (r = 0.82; p = 0.001). Protein binding of aspirin was estimated at 58.3% +/- 9.6% by in vivo ultrafiltration and could not be estimated by in vitro ultracentrifugation because the concentration of unbound aspirin in plasma was below the limit of detection for the assay. CONCLUSION: In vivo ultrafiltration can be used to measure protein binding of drugs and has potential advantages over conventional methods. A sensitive assay may not be required because the unbound drug need not be measured, measurement in vivo may maintain more physiologic conditions, and it may be useful in measuring protein binding of drugs that are degraded rapidly in vitro.

Association of panic disorder and panic attacks with hypertension.

PURPOSE: Previous studies of the association between hypertension and panic disorder were uncontrolled or involved small numbers of patients. PATIENTS AND METHODS: We compared the prevalence of panic disorder and panic attacks in 351 patients with documented hypertension who were randomly selected from all hypertensive patients registered in one primary care practice with age- and gender-matched normotensive patients from the same practice and with hypertensive patients attending a hospital clinic. All three groups completed questionnaires for panic disorder based on standard criteria, as well as the Hospital Anxiety and Depression scale. RESULTS: The prevalence of current (previous 6 months) panic attacks was significantly greater in primary care patients with hypertension (17%, P <0.05) and hospital-based hypertensive patients (19%, P <0.01) than in normotensive patients (11%). Similar results were seen for lifetime panic attacks (35% versus 39% versus 22%; both P for comparisons with normotensive patients <0.001). The prevalence of panic disorder was significantly greater in primary care patients with hypertension (13%) than normotensive patients (8%, P <0.05). Anxiety scores were significantly higher in both hypertensive groups than in normotensive patients. Depression scores were significantly higher in hospital-based hypertensive patients than in the other two groups. The reported diagnosis of hypertension antedated the onset of panic attacks in a large majority of patients (P <0.01). CONCLUSIONS: Physicians caring for patients with hypertension should be aware of the significantly greater prevalence of panic attacks in these patients.

Is coronary risk an accurate surrogate for cardiovascular risk for treatment decisions in mild hypertension? A population validation.

OBJECTIVE: To examine the relationship between coronary (CHD) and cardiovascular (CVD) risk in patients with uncomplicated mild hypertension and to determine the accuracy of using CHD risk > or = 15% over 10 years to identify for antihypertensive treatment those patients with CVD risk > or = 20% over 10 years as advised in recent British guidelines. DESIGN: Comparison of decisions made using CHD risk > or = 15% over 10 years calculated by the Framingham risk function and estimated using a simple table with CVD risk > or = 20% over 10 years. SETTING: British population. SUBJECTS: People aged 35-64 years with uncomplicated mild systolic hypertension (SBP 140-159 mmHg, n = 624) from the 1995 Scottish Health Survey. MAIN OUTCOME MEASURES: Relationship between CHD and CVD risk. Sensitivity, specificity, positive and negative predictive values (PPV and NPV). RESULTS: CHD risk 15% over 10 years was equivalent to CVD risk 21% over 10 years. Exact CHD risk > or = 15% over 10 years had sensitivity 79%, specificity 98%, PPV 94% and NPV 93% in detecting CVD risk > or = 20% over 10 years. Use of the table to estimate CHD risk > or = 15% over 10 years gave sensitivity 88%, specificity 90%, PPV 76% and NPV 95%. CONCLUSION: CHD risk appears acceptably accurate for targeting treatment in mild hypertension. The risk assessment table, which slightly overestimates CHD risk, was more sensitive in identifying patients with CVD risk > or = 20% over 10 years and may be preferable to using exact CHD risk. European guidelines which suggest targeting treatment for mild hypertension at CHD risk > or = 20% over 10 years are over-conservative compared with British guidelines.

Implication of recent trials with b-hydroxy-b-methylglutaryl coenzyme A reductase inhibitors for hypertension management.

BACKGROUND: There is broad agreement that statin treatment should be targeted at absolute coronary heart disease (CHD) risk but no consensus on the level of risk to target. We have examined the implications of adopting three different treatment policies for the management of hypertensive patients in the UK using data from treated hypertensives aged 35-69 years included in the Health Survey for England (1993). METHODS: We calculated the proportion of hypertensive patients with existing atherosclerotic cardiovascular disease requiring statin treatment for secondary prevention of CHD. For those without atherosclerotic cardiovascular disease (primary prevention), we estimated CHD risk from the Framingham equation and examined the proportion with CHD risk exceeding thresholds of 4.5, 3 and 1.5% per year. RESULTS: Twenty-one percent of treated hypertensives would require statin treatment for secondary prevention of CHD. When the CHD event threshold for statin treatment was set at > or =4.5% per year [equivalent to a number needed to treat (NNT) in 5 years of 13] a further 0.6% of hypertensive patients were identified for treatment; at a threshold of 3.0% per year (NNT = 20) 5.5% of patients were identified for primary prevention; and at a threshold of 1.5% per year (NNT = 40) 28.5% of patients were identified for primary prevention. CONCLUSIONS: Those needing secondary prevention are first priority for statins and 21% of hypertensive patients will require treatment Formulation of guidelines for primary prevention should take into account the NNT; the proportion of patients targeted for treatment; the cost-effectiveness and the total cost of treatment. Current British guidance will entail treating an additional 5.5% of hypertensive patients for primary prevention and therefore 27% of hypertensive patients.

Panic disorder, anxiety and depression in resistant hypertension--a case-control study.

BACKGROUND: It has been suggested that panic disorder can cause or contribute to hypertension or resistance to antihypertensive drugs. OBJECTIVE: To compare the prevalences of panic disorder, panic attacks, anxiety and depression between patients with resistant hypertension and age- and sex-matched patients with non-resistant hypertension. DESIGN: A case-control study of patients attending the Sheffield Hypertension Clinic, using self-completed postal questionnaires to assess panic disorder, anxiety and depression. PATIENTS CASES: With resistant hypertension were defined as patients who presently or previously had systolic blood pressure above 160 mmHg or diastolic blood pressure above 90 mmHg despite the use of three or more antihypertensive agents at full dose. For each of 136 cases, one control with non-resistant hypertension, defined as controlled to < or = 160/90 mmHg by one or two antihypertensive agents, was identified by a bias-free method. Cases and controls were matched for age and sex. MAIN OUTCOME MEASURES: Lifetime and current prevalence of panic attacks, the prevalences of panic disorder, anxiety and depression by Hospital Anxiety and Depression Scale scores, and the severity and frequency of panic attacks. RESULTS: Of the resistant hypertensive patients, 33% had experienced a panic attack compared with 39% of the control non-resistant hypertensives (resistant-non-resistant -6%, 95% confidence interval -19 to +7%). Twelve per cent of the resistant patients and 14% of controls fulfilled the criteria for a current or previous diagnosis of panic disorder (resistant-non-resistant -2%, 95% confidence interval -11% to +7%). There were also no significant differences between the groups in the prevalences of current panic attacks, panic attacks rated as moderate or worse, spontaneous panic attacks and in the frequency of panic attacks. There remained no significant difference between the groups for panic attacks and panic disorder when the analysis was limited to those patients who had idiopathic hypertension. The two groups did not differ significantly in scores for anxiety and depression measured by the Hospital Anxiety and Depression Scale. CONCLUSION: We observed no differences in the prevalences of panic, anxiety and depression between patients with resistant hypertension and non-resistant controls. These factors are probably not implicated in resistance to drug treatment. However, the prevalences of panic disorder and panic attacks were remarkably high in both groups of patients attending a hospital hypertension clinic. The relationship between panic disorder and hypertension deserves further study in a general hypertensive population.

Nephrotoxicity of parenterally administered cyclosporine after orthotopic liver transplantation.

The frequency of nephrotoxicity in the absence of identifiable prerenal or postrenal causes within five days of operation in 27 liver transplant recipients was found to be 71% in those treated with i.v. cyclosporine alone, 37.5% with i.m. cyclosporine alone, and 16.7% with prednisolone and azathioprine. Renal failure following i.v. cyclosporine was characterized by an immediate fall in urine output and creatinine clearance with well-preserved tubular function--findings consistent with a reduction in renal blood flow or glomerular filtration rate, or both.

Adult respiratory distress syndrome and convulsions associated with administration of cyclosporine in liver transplant recipients.

A "capillary leak" syndrome resulting from cyclosporine-induced membrane toxicity has been postulated as the cause of convulsions and pulmonary edema in bone marrow transplant recipients. We describe here the occurrence of similar complications in a group of 21 adults receiving liver transplants since July 1982. Of 12 patients treated with i.v. cyclosporine (4 mg/kg/day), 2 developed an adult respiratory distress syndrome (ARDS) within five days of the operation, but it was not found in those given prednisolone (0.05-1.0 mg/kg/day) and azathioprine (1.0 mg/kg/day). ARDS only occurred when cyclosporine was administered through a central vein, and therefore might be related to high concentrations of cyclosporine reaching the pulmonary circulation and causing damage to vascular membranes. Convulsions occurred in one patient given i.v. cyclosporine, and in three when therapy was changed and cyclosporine and corticosteroids were used in combination. Convulsions did not occur inthe same patients as ARDS, were not part of a generalized "capillary leak" syndrome, and were not associated with hypertension or renal failure, as reported elsewhere in children. Fluid retention consequent on cyclosporine administration aggravated by the use of corticosteroids appears to be the most likely explanation of the convulsions.

Enantioselective and diastereoselective aspects of the oxidative metabolism of metoprolol.

Enantio- and diastereoselective aspects of oxidative metabolism of metoprolol (1) were examined in the presence of rat liver and human liver microsomes using a pseudoracemate of 1, made up of equal molar (2R)-1-d0 and (2S)-1-d2, as substrate. Both O-demethylation and alpha-hydroxylation showed only slight enantioselectivity, 2R/2S ratios being 1.18 and 0.93 for these pathways in rat liver microsomes and 1.09 and 0.92 in human liver microsomes. In the presence of the rat liver microsomal fraction, alpha-hydroxylation yielded predominantly the 1'R-hydroxy product, 1'R/1'S ratio greater than 12, regardless of the stereochemistry of the side chain. In humans (extensive metabolizers) administered a single 50 mg oral dose of pseudoracemic metoprolol tartrate, urinary alpha-hydroxymetoprolol (2) accounted for 9.3 +/- 2.4% of the dose, 2R/2S ratio 0.85 +/- 0.14, and the carboxylic acid metabolite 4, accounted for 52.7 +/- 6.8% of the dose, 2R/2S ratio 1.15 +/- 0.09. The data suggested that preferential O-demethylation of the (2R)-enantiomer of 1 could contribute to the 2S greater than 2R plasma ratio of metoprolol enantiomers observed in this population.

Effect of isoniazid administration on selected rat and mouse hepatic microsomal mixed-function oxidases and in vitro [14C]acetylhydrazine-derived covalent binding.

The effect of isoniazid on selected microsomal mixed-function oxidase activities and on the microsomal metabolism of its own metabolite, acetylhydrazine, to a highly reactive compound which covalently binds to intracellular macromolecules was characterized in male C57BL6 mice and male Sprague-Dawley rats. In comparison with controls, isoniazid pretreatment of rats significantly increased the sp. act. of acetanilide 4-hydroxylase and the in vitro [14C]acetylhydrazine-derived covalent binding to hepatic microsomes but significantly decreased the sp. act. of benzo[a]pyrene hydroxylase and testosterone 16 alpha-hydroxylase. Isoniazid treatment of mice had no effect on any of these parameters except for a significant reduction in sp. act. of testosterone 7 alpha-hydroxylase. Thus the pathway of isoniazid metabolism leading to the formation of reactive metabolites of acetylhydrazine is enhanced by isoniazid pretreatment in rats but not in mice. The presence of similar routes of isoniazid metabolism in man may account for the 8.7-24% incidence of subclinical hepatocellular damage observed in patients receiving isoniazid alone in the chemoprophylaxis of tuberculosis.

The rationale for differing national recommendations for the treatment of hypertension.

This article examines the rationale for the differences in the guidelines for hypertension management of four national or international bodies: the Joint National Committee (JNC-V), The World Health Organization/International Society of Hypertension (WHO-ISH), the British Hypertension Society (BHS), and the New Zealand guidelines. These guidelines agree on many aspects of management, but differ on two very important points-the drugs of first choice for hypertension, and the indications for drug treatment of uncomplicated mild hypertension. JNC-V recommends treatment routinely of all people with a sustained blood pressure of 140/90 mm Hg, whereas the BHS guidelines advise treatment routinely at 160/100 mm Hg. Such differences in the threshold for treatment have a major impact on the proportion of the adult population to be treated, and on the benefit from treatment. JNC-V was heavily influenced by the Hypertension Detection and Follow-up Program (HDFP), which appeared to show a large benefit from the treatment of uncomplicated mild hypertension, whereas the BHS guidelines were influenced by the Medical Research Council (MRC) Trial, which showed a very small benefit. However, the apparent differences in absolute benefit between these, and other, randomized controlled trials is related entirely to differences in the absolute cardiovascular risk of the populations studied. In populations and in individual patients the benefit from antihypertensive treatment is determined by the absolute cardiovascular risk. Blood pressure by itself is a very weak predictor of risk or benefit from treatment. In uncomplicated mild hypertension the need for drug therapy should be based on the absolute risk of cardiovascular complications, estimated by considering age, sex, serum cholesterol level, diabetes mellitus status, and smoking habits, in addition to blood pressure. Doctors cannot estimate absolute risk accurately informally or intuitively, and the next generation of guidelines should incorporate a simple but accurate method for estimating cardiovascular risk, similar to that in the New Zealand guidelines. The decision to treat, or not treat, uncomplicated mild hypertension should be based on a formal estimate of absolute cardiovascular risk and not on an arbitrary blood pressure threshold. As regards drugs of first choice, the available evidence supports strongly the stance of JNC-V and JNC VI that diuretics and beta-blockers should be preferred unless they are contraindicated, or unless there are positive indications for other drug classes.

Identification of pathogenic Leishmania promastigotes by DNA: DNA hybridization with kinetoplast DNA cloned into E. coli plasmids.

We report the characterization of Leishmania (L. infantum, L. donovani, and L. major) kinetoplast DNA (kDNA) by the use of restriction endonuclease digestion patterns and Southern hybridizations. Overall, the sizes and fragment patterns of MspI restriction endonuclease-produced DNA fragments vary from species to species. However, kDNA isolates from different species and strains cross-reacted to a great extent in Southern hybridization experiments. Only kDNA isolated from L. infantum and L. major had little homology during hybridization reactions. To prepare DNA probes that would differentiate between species of Leishmania, minicircle kDNA was digested with restriction enzymes and ligated to an E. coli plasmid. Several plasmids were isolated that specifically detect in hybridization experiments as few as 5 X 10(3) L. donovani or L. infantum promastigotes lysed on nitrocellulose filters.

Trypanosoma brucei rhodesiense: enhancement of infection rates in the tsetse fly, Glossina morsitans, by feeding artificial bloodmeal mixtures.

Low mature salivary gland (SG) infection rates (6%) in less than 24-hour-old flies fed on blood containing bloodform trypanosomes can be significantly enhanced by feeding flies an artificial mixture containing procyclic forms in a red cell: culture medium mixture (procyclic mixture, SG rate = 21.0%). However, enhancement is not solely a function of the use of procyclic forms since blood forms fed to flies in the same red cell: culture medium mixture produce SG rates (15.4%) intermediate to those of blood forms in blood and procyclic mixtures. Use of these artificial mixtures produces a similar result in 24- to 48-hour-old flies and also tends to equalize their infection rates with those found in less than 24-hour-old flies. The possible relationships between the different infection rates observed and digestive proteinases in the tsetse fly are discussed.

Restriction endonuclease analysis of Leishmania kinetoplast DNA characterizes parasites responsible for visceral and cutaneous disease.

The kinetoplast DNA (kDNA) from promastigotes of Leishmania responsible for Old and New World cutaneous and visceral disease was characterized to determine if species and strains causing similar or different diseases could be identified. Restriction enzymes were used to digest kDNA into fragments that were separated into characteristic banding patterns after electrophoresis in agarose or linear gradient polyacrylamide gels. Hybridization was conducted with a 32P-kDNA probe and kDNA fragments transferred from agarose gels to nitrocellulose paper. Leishmania species causing cutaneous diseases in the New and Old Worlds all had different kDNA digest patterns. Visceralizing Leishmania from the New and Old Worlds also had different kDNA restriction fragment patterns although Leishmania donovani parasites with similar fragment patterns were isolated from several humans from central Kenya. Nucleotide sequences were shared among kDNA networks from L. donovani, Leishmania d. chagasi, Leishmania d. infantum, Leishmania tropica, and Leishmania major as determined by hybridization with a 32P-kDNA probe from L. donovani. However, no hybridization was detected between the L. donovani 32P-kDNA probe and kDNA from Leishmania aethiopica or Leishmania braziliensis panamensis. Leishmania characterization results for the same isolates from the published literature were compared and kinetoplast DNA analysis was found to be one of the most sensitive procedures for species and strain identification.

Prediction of coronary risk for primary prevention of coronary heart disease: a comparison of methods.

Most recent guidelines advise targeting of lipid lowering for primary prevention at those at high absolute coronary (CHD) risk. We compared the accuracy of five CHD risk assessment methods in identifying such patients: one based on total cholesterol > or = 6.5 mmol/l plus two risk factors, and four based on the Framingham risk function (the European Task Force chart and Sheffield table, both using total cholesterol and the New Zealand chart and modified Sheffield table, both using total: HDL cholesterol ratio) for predicting CHD event risk > or = 2% per year, calculated by an independent risk function, PROCAM, in 126 treated hypertensive men. Cholesterol threshold plus two risk factors had sensitivity 59% and specificity 63%, did not identify some very high-risk patients, and identified very low-risk patients. Framingham-based methods using total cholesterol alone had sensitivity 90-98% and specificity 37-43%, and identified high-risk patients well, but identified some patients at very low risk. Methods based on total: HDL cholesterol ratio had sensitivity 90-98% and specificity 60-63%, and did not identify incorrectly patients at very low CHD risk. Methods based on cholesterol threshold and counting of risk factors are too inaccurate for targeting drug therapy for primary prevention of CHD. Framingham-based methods should incorporate HDL-cholesterol as the total: HDL cholesterol ratio.