Towards an international language for incontinence-associated dermatitis (IAD): design and evaluation of psychometric properties of the Ghent Global IAD Categorization Tool (GLOBIAD) in 30 countries.

BACKGROUND: Incontinence-associated dermatitis (IAD) is a specific type of irritant contact dermatitis with different severity levels. An internationally accepted instrument to assess the severity of IAD in adults, with established diagnostic accuracy, agreement and reliability, is needed to support clinical practice and research. OBJECTIVES: To design the Ghent Global IAD Categorization Tool (GLOBIAD) and evaluate its psychometric properties. METHODS: The design was based on expert consultation using a three-round Delphi procedure with 34 experts from 13 countries. The instrument was tested using IAD photographs, which reflected different severity levels, in a sample of 823 healthcare professionals from 30 countries. Measures for diagnostic accuracy (sensitivity and specificity), agreement, interrater reliability (multirater Fleiss kappa) and intrarater reliability (Cohen's kappa) were assessed. RESULTS: The GLOBIAD consists of two categories based on the presence of persistent redness (category 1) and skin loss (category 2), both of which are subdivided based on the presence of clinical signs of infection. The agreement for differentiating between category 1 and category 2 was 0·86 [95% confidence interval (CI) 0·86-0·87], with a sensitivity of 90% and a specificity of 84%. The overall agreement was 0·55 (95% CI 0·55-0·56). The Fleiss kappa for differentiating between category 1 and category 2 was 0·65 (95% CI 0·65-0·65). The overall Fleiss kappa was 0·41 (95% CI 0·41-0·41). The Cohen's kappa for differentiating between category 1 and category 2 was 0·76 (95% CI 0·75-0·77). The overall Cohen's kappa was 0·61 (95% CI 0·59-0·62). CONCLUSIONS: The development of the GLOBIAD is a major step towards a better systematic assessment of IAD in clinical practice and research worldwide. However, further validation is needed.

Competition in the Dutch hospital sector: an analysis of health care volume and cost.

This paper evaluates the impact of market competition on health care volume and cost. At the start of 2005, the financing system of Dutch hospitals started to be gradually changed from a closed-end budgeting system to a non-regulated price competitive prospective reimbursement system. The gradual implementation of price competition is a 'natural experiment' that provides a unique opportunity to analyze the effects of market competition on hospital behavior. We have access to a unique database, which contains hospital discharge data of diagnosis treatment combinations (DBCs) of individual patients, including detailed care activities. Difference-in-difference estimates show that the implementation of market-based competition leads to relatively lower total costs, production volume and number of activities overall. Difference-in-difference estimates on treatment level show that the average costs for outpatient DBCs decreased due to a decrease in the number of activities per DBC. The introduction of market competition led to an increase of average costs of inpatient DBCs. Since both volume and number of activities have not changed significantly, we conclude that the cost increase is likely the result of more expensive activities. A possible explanation for our finding is that hospitals look for possible efficiency improvements in predominantly outpatient care products that are relatively straightforward, using easily analyzable technologies. The effects of competition on average cost and the relative shares of inpatient and outpatient treatments on specialty level are significant but contrary for cardiology and orthopedics, suggesting that specialties react differently to competitive incentives.

Surface-tension driven open microfluidic platform for hanging droplet culture.

The hanging droplet technique for three-dimensional tissue culture has been used for decades in biology labs, with the core technology remaining relatively unchanged. Recently microscale approaches have expanded the capabilities of the hanging droplet method, making it more user-friendly. We present a spontaneously driven, open hanging droplet culture platform to address many limitations of current platforms. Our platform makes use of two interconnected hanging droplet wells, a larger well where cells are cultured and a smaller well for user interface via a pipette. The two-well system results in lower shear stress in the culture well during fluid exchange, enabling shear sensitive or non-adherent cells to be cultured in a droplet. The ability to perform fluid exchanges in-droplet enables long-term culture, treatment, and characterization without disruption of the culture. The open well format of the platform was utilized to perform time-dependent coculture, enabling culture configurations with bone tissue scaffolds and cells grown in suspension. The open nature of the system allowed the direct addition or removal of tissue over the course of an experiment, manipulations that would be impractical in other microfluidic or hanging droplet culture platforms.

National survey on the natural radioactivity and 222Rn exhalation rate of building materials in The Netherlands.

The present study reports on results of a nation-wide survey on the natural radioactivity concentrations and Rn exhalation rates of the prevailing building materials in the Netherlands. In total 100 samples were taken and analyzed for the activity concentrations of Ra, Ra, Th, and K and for their Rn exhalation rate. The sampled materials consisted of gypsum products, aerated concrete, sand-lime and clay bricks, mortars and concrete, representing about 95% of the stony building materials used in the construction of Dutch homes. The laboratory analyses were performed according to two well-documented standard procedures, the interlaboratory reproducibility of which is found to be within 5% on average. The highest radionuclide concentrations were found in a porous inner wall brick to which fly ash was added. The second highest were clay bricks with average Ra and Ra levels around 40 Bq kg. Concrete and mortar show the highest exhalation rates with a fairly broad range of 1 to 13 microBq (kg s). Low natural radioactivity levels are associated with either natural gypsum (products) or gypsum from flue gas desulphurization units, and low exhalation rates with clay bricks. To evaluate the radiological impact the radioactivity concentrations in each sample were combined into a so-called dose factor, representing the absorbed dose rate in a room with a floor, walls and ceiling of 20 cm of the material in question. For that purpose, calculations with the computer codes MCNP, Marmer and MicroShield on the specific absorbed dose rates were incorporated in the paper. The results of these codes corresponded within 6% and average values were calculated at 0.90, 1.10, and 0.080 nGy h per Bq kg for the U series, the Th series, and K, respectively. Model calculations on the external dose rate, based on the incidence of the various building materials in 1,336 living rooms, are in accordance with measured data.

[11C]methionine PET, histopathology, and survival in primary brain tumors and recurrence.

BACKGROUND AND PURPOSE: [(11)C]Methionine (MET) PET imaging is a sensitive technique for visualizing primary brain tumors and recurrence/progression after therapy. The aim of this study was to evaluate the relationship between the uptake of MET and histopathologic grading and to investigate the prognostic value of the tracer, in both settings. METHODS: Cerebral uptake of MET was determined in 52 patients: in 26 patients for primary staging (group A) and 26 patients with suspected brain tumor recurrence/progression after therapy (group B). Semiquantitative methionine uptake indices (UI) defined by the tumor (maximum)-to-background ratio was correlated with tumor grade and final outcome. RESULTS: Overall median survival was 34.9 months. MET showed pathologically increased uptake in 41 of 52 scans. Although a weak linear correlation between MET uptake and grading was observed (R = 0.38, P = .028), analysis of variance showed no significant differences in MET UI between tumor grades for either group A or B. Benign and grade I lesions showed significant difference in MET uptake in comparison with higher grade lesions (P = .006). Using Kaplan-Meier survival analysis, no thresholds could be found at which MET was predictive for survival. Proportional hazard regression showed that only WHO grading class (low versus high) was predictive of survival (P = .015). CONCLUSION: Interindividual MET uptake variability does not allow noninvasive grading on an individual patient basis. Moreover, there is no significant prognostic value in studying maximal methionine UI in brain tumors. The clinical use of MET should therefore be primarily focused on questions such as detection of recurrence, biopsy guidance, and radiation therapy target volume delineation.

Comparison between pathogen directed antibiotic treatment and empirical broad spectrum antibiotic treatment in patients with community acquired pneumonia: a prospective randomised study.

BACKGROUND: There is much controversy about the ideal approach to the management of community acquired pneumonia (CAP). Recommendations differ from a pathogen directed approach to an empirical strategy with broad spectrum antibiotics. METHODS: In a prospective randomised open study performed between 1998 and 2000, a pathogen directed treatment (PDT) approach was compared with an empirical broad spectrum antibiotic treatment (EAT) strategy according to the ATS guidelines of 1993 in 262 hospitalised patients with CAP. Clinical efficacy was primarily determined by the length of hospital stay (LOS). Secondary outcome parameters for clinical efficacy were assessment of therapeutic failure on antibiotics, 30 day mortality, duration of antibiotic treatment, resolution of fever, side effects, and quality of life. RESULTS: Three hundred and three patients were enrolled in the study; 41 were excluded, leaving 262 with results available for analysis. No significant differences were found between the two treatment groups in LOS, 30 day mortality, clinical failure, or resolution of fever. Side effects, although they did not have a significant influence on the outcome parameters, occurred more frequently in patients in the EAT group than in those in the PDT group (60% v 17%, 95% CI -0.5 to -0.3; p<0.001). CONCLUSIONS: An EAT strategy with broad spectrum antibiotics for the management of hospitalised patients with CAP has comparable clinical efficacy to a PDT approach.

Value of intensive diagnostic microbiological investigation in low- and high-risk patients with community-acquired pneumonia.

In a prospective study to evaluate the diagnostic yield of different microbiological tests in hospitalised patients with community-acquired pneumonia, material for microbiological investigation was obtained from 262 patients. Clinical samples consisted of the following: sputum for Gram staining, culture, and detection of pneumococcal antigen; blood for culture and serological tests; urine for detection of Legionella pneumophila serogroup 1 antigen and pneumococcal antigen; and specimens obtained by fiberoptic bronchoscopy. A pathogen was identified in 158 (60%) patients, with Streptococcus pneumoniae (n=97) being the most common causative agent of community-acquired pneumonia. In 82% of the 44 patients with an adequate sputum specimen, a positive Gram stain was confirmed by positive sputum culture. S. pneumoniae infections were detected principally when adequate sputum specimens were examined by Gram stain and culture and when adequate and inadequate sputum specimens were tested for the presence of pneumococcal antigen (n=58; 60%). The urinary pneumococcal antigen test was the most valuable single test for detection of S. pneumoniae infections (n=52; 54%) when sputum pneumococcal antigen determination was not performed. Fiberoptic bronchoscopy was of additive diagnostic value in 49% of the patients who did not expectorate sputum and in 52% of those in whom treatment failed. Investigation of sputum by a combination of Gram stain, culture, and detection of pneumococcal antigen was the most useful means of establishing an aetiological diagnosis of community-acquired pneumonia, followed by testing of urine for pneumococcal antigen. Fiberoptic bronchoscopy may be of additional value when treatment failure occurs.

RP-HPLC separation of the diastereomers of technetium-99m labelled tropanes and identity confirmation using radio-LC-MS.

99mTc-TRODAT-1 (technetium(V)-oxo-2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]oct-2-yl]methyl](2-mercaptoethyl)amino]ethyl]]amino]-ethanethiolato(3-)) and 99mTc-TRODAT-M, the 4-methylphenyl derivative of 99mTc-TRODAT-1, are at this moment being evaluated in clinical trials as imaging agents for the central nervous dopamine transporter system. Both compounds are formed as a mixture of two major diastereomers. As the tracer concentration in preparations for clinical investigations is very low (30-150 pmol/ml), identification of these 99mTc-complexes was, up to now, carried out indirectly using X-ray diffraction analysis of the corresponding rhenium complexes which can be synthesized in gram amounts. In this study, we developed a convenient and practical reversed phase HPLC-method for purification and isolation of the respective diastereomers of 99mTc-TRODAT-1 and three of its derivatives using mixtures of solvents which are compatible with biological studies, i.e. aqueous buffers and ethanol. Furthermore, direct identity confirmation of the 99mTc-complexes using radio-LC-MS was successfully elaborated.

Molecular genetic evidence for parthenogenesis in the Burmese python, Python molurus bivittatus.

Parthenogenesis among reptiles is rare. Only a few species have the ability to reproduce asexually. Most of these are obligate parthenogenetic species that consist (almost) entirely of females, which can reproduce solely through parthenogenesis. Rarer are sexual species that only sporadically reproduce through parthenogenesis. A female Python molurus bivittatus (Reptilia, Boidae) from the Artis Zoo, Amsterdam, produced eggs in five consecutive years that contained embryos while she was isolated from males. These eggs might be fertilized with stored sperm, or might be the product of parthenogenesis. Parthenogenesis has not been shown for the Boidae family before. We performed parentship analyses on the snake and seven of her embryos using microsatellites and AFLP. Four microsatellite loci developed for this species combined with three loci developed previously for different snake species revealed too little variation to discriminate between sperm retention and parthenogenesis. With AFLP we were able to confirm that the Artis Zoo female reproduced parthenogenetically. Because the offspring are genetically identical to their mother, whereas in previous studies on sporadic parthenogenesis in snakes a loss of genetic information was reported, we conclude that the meiotic pathways that produce the diploid egg cells are different.

Early restaging positron emission tomography with ( 18)F-fluorodeoxyglucose predicts outcome in patients with aggressive non-Hodgkin's lymphoma.

BACKGROUND: Less than half of all patients with aggressive non-Hodgkin's lymphoma (NHL) are cured with standard chemotherapy. Therefore, it is important to distinguish between responders to standard treatment and non-responders who may benefit from an early change to a more effective therapy. This study was intended to assess the value of a midtreatment fluorine-18 fluorodeoxyglucose positron emission tomography ([(18)F]FDG-PET) scan to predict clinical outcome in patients with aggressive NHL. PATIENTS AND METHODS: Seventy newly diagnosed patients with aggressive NHL, who were treated with doxorubicin-containing chemotherapy, underwent a [(18)F]FDG-PET scan at midtreatment. Presence or absence of abnormal [(18)F]FDG uptake was related to progression-free survival (PFS) and overall survival (OS) using Kaplan-Meier survival analysis. Multivariate analysis was performed to evaluate the effect of the International Prognostic Index (IPI) and early [(18)F]FDG-PET findings on PFS and OS. RESULTS: At midtreatment, 33 patients showed persistent abnormal [(18)F]FDG uptake and none of these patients achieved a durable complete remission (CR), whereas 37 patients showed a negative scan; 31/37 remained in CR, with a median follow-up of 1107 days. Only 6/37 patients either achieved a partial response or relapsed. Comparison between groups indicated a statistically significant association between [(18)F]FDG-PET findings and PFS (P <1 x 10(-5)) and OS (P <1 x 10(-5)). In multivariate analysis, [(18)F]FDG-PET at midtreatment was a stronger prognostic factor for PFS (P <1 x 10(-7)) and OS (P <9 x 10(-6)) than the IPI (P <0.11 and P <0.03, respectively). CONCLUSIONS: Early restaging [(18)F]FDG-PET may be used to tailor induction chemotherapy in patients with aggressive NHL.

[Dental complaints are found always between the ears. Somatization in the dental practice]. / Een tandheelkundige klacht zit altijd tussen de oren. Somatisatie in de tandartspraktijk.

The aim of the present study was to investigate the occurrence of somatization-specific behaviour in the dental setting and its relationship with patients' report of both dental and psychological complaints. Somatization-specific behaviour was operationalized as an unexplained dental complaint, high dental attendance, a high treatment consumption or an unreasonable demand with regard to treatment. Of the 309 patients 8.7% fulfilled the criteria for somatization-specific behaviour, which was mainly expressed as a high frequency of attendance (6.8%). Women showed significantly more often (73%) somatization-specific behaviour than men (27%). Further, a relation between depression and somatization-specific behaviour was found. Particularly patients with a relatively high level of long-lasting dental complaints demonstrated somatization-specific behaviour.

Evaluation of 99mTc-MAMA-chrysamine G as an in vivo probe for amyloidosis.

To date, systemic amyloidosis is diagnosed histologically using Congo red staining or in vivo using iodine-123 labelled serum amyloid P component (123I-SAP) scintigraphy. We developed 99mTc-MAMA-CG, a 99mTc-labelled derivative of the lipophilic Congo red analogue chrysamine G (CG), as a possible alternative to 123I-SAP. In vivo 99mTc-MAMA-CG scintigraphy, performed in chickens with spontaneous joint amyloidosis, resulted as soon as 10 min after injection in scintigraphic images showing uptake of activity in amyloid-loaded organs (liver, joints). One of these chickens was studied also with 123I-SAP resulting in scintigraphic images revealing 123I-SAP binding to amyloid deposits in the liver. However, up to 11 h after injection no radioactivity was visible in the amyloid positive joints. In vitro autoradiography, performed on sections of chicken joints with Enterococcus faecalis induced amyloid arthropathy (chjAA), demonstrated the failure of 99mTc-MAMA-CG to bind significantly to amyloid deposits in the presence of 10 microM Congo red The specificity of 99mTc-MAMA-CG localisation was also established by the absence of 99mTc-MAMA-CG binding in non-amyloidotic organs in vitro and in vivo. 99mTc-MAMA-CG did not show any sign of acute toxicity. These findings establish the usefulness of 99mTc-MAMA-CG as a non-invasive in vivo diagnostic probe in chickens with amyloid arthropathy and suggest that it may also be applicable to human amyloidosis.

In vitro affinity of 99Tcm-labelled N2S2 conjugates of chrysamine G for amyloid deposits of systemic amyloidosis.

To date, systemic amyloidosis is diagnosed histologically in vitro using Congo red staining or in vivo using iodine-123 serum amyloid P component (123I-SAP) scintigraphy. 99Tcm-labelled derivatives of chrysamine G (CG), a lipophilic analogue of Congo red, were synthesized as potential tracer agents for direct and quantitative scintigraphic evaluation of amyloid deposits. To determine the affinity of 99Tcm-MAMA-CG, 99Tcm-Me4MAMA-CG and 99Tcm-MAMA-CG diethyl ester for amyloid, in vitro autoradiography was performed on sections of human kidney biopsy cylinders from kidneys with amyloid deposits (types AA, Alambda and Akappa) or control kidney tissue after incubation with the respective tracer agents. The binding of 99Tcm-MAMA-CG and its tetramethyl derivative was higher to kidney biopsy material with amyloid deposits of the AA, Alambda or Akappa type compared with control kidney tissue. This higher binding was prevented by the presence of 10 microM Congo red in the incubation medium. The diethyl ester of 9Tcm-MAMA-CG did not demonstrate increased binding to Congo red-positive kidney tissue. In conclusion, 99Tcm-MAMA-CG and 99Tcm-Me4MAMA-CG localize specifically to amyloid deposits in human kidney tissue, suggesting that these tracer agents may be applicable as specific targeting agents for diagnostic purposes in clinical amyloidosis.

PET reversed mismatch in an experimental model of subacute myocardial infarction.

The aim of this study was to evaluate the relationship between flow/metabolism, histology and functional follow-up in a sheep model of subacute myocardial infarction. In eight juvenile sheep, a myocardial infarction was induced by intracoronary injection of macrobeads. Left ventricular function was evaluated using echocardiography. 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG)/nitrogen-13-labelled ammonia (13NH3) positron emission tomography (PET) was performed at 6 weeks and 16 weeks after embolization. In five sheep, a dynamic carbon-11 acetate study was performed. In each animal, two regions of interest were defined on the polar map, corresponding to the embolized and the non-embolized region. After the final measurements, the hearts were processed for histological evaluation. PET revealed a moderately decreased flow and oxidative metabolism in the embolized region at 6 weeks, without significant changes at follow-up. At 6 weeks, 18F-FDG uptake in the embolized area was more severely decreased as compared to the flow index in the embolized area (P < 0.05). At 16 weeks, 18F-FDG metabolism had significantly recovered (P < 0.05). Serial echocardiography showed a persistent decrease in global and regional left ventricular function. Histology revealed a mix of micro-infarcted and viable tissue in the embolized region. In this model of subacute myocardial infarction, a PET "reversed mismatch" pattern was observed, with partial recovery of 18F-FDG uptake at follow-up. The histological counterpart of this PET pattern appears to be patchy necrosis.

99mTc-MAMA-chrysamine G, a probe for beta-amyloid protein of Alzheimer's disease.

Chrysamine G (CG), an analogue of Congo red, is known to bind in vitro to the beta-amyloid protein (Abeta 10-43) and to homogenates of several regions of the brain of Alzheimer's disease (AD) patients. We synthesised a conjugate of 2-(acetamido)-CG with a bis-S-trityl protected monoamide-monoaminedithiol (MAMA-Tr(2)) tetraligand, which was efficiently deprotected and labelled with a 75% yield with technetium-99m, to obtain (99m)Tc-MAMA-CG. In mice, (99m)Tc-MAMA-CG was cleared mainly by the hepatobiliary system, resulting in a fast blood clearance. Brain uptake of (99m)Tc-MAMA-CG was low. Co-injection with the blood pool tracer iodine-125 human serum albumin ((125)I-HSA) demonstrated a brain/blood activity ratio for (99m)Tc-MAMA-CG that was significantly higher than that for (125)I-HSA (t test for dependent samples, P<0.02), indicating the ability of (99m)Tc-MAMA-CG to cross the blood-brain barrier. In vitro autoradiography demonstrated pronounced binding of (99m)Tc-MAMA-CG to beta-amyloid deposits in autopsy sections of the parietal and occipital cortex of an AD patient as compared with controls. Adding 10 microM Congo red during incubation displaced the binding of (99m)Tc-MAMA-CG. Congo red staining and autoradiography identified the same lesions. (99m)Tc-MAMA-CG seems to bind selectively to beta-amyloid deposition in human brain parenchyma and blood vessels in vitro and thus might be a lead compound for further development of a useful tracer agent for the in vivo diagnosis of Alzheimer's disease.

Worldwide evaluation of DNA sequencing approaches for identification of drug resistance mutations in the human immunodeficiency virus type 1 reverse transcriptase.

A panel (ENVA-1) of well-defined blinded samples containing wild-type and mutant human immunodeficiency virus type 1 (HIV-1) reverse transcriptase was analyzed by automated DNA sequencing in 23 laboratories worldwide. Drug resistance mutations at codons 41, 215, and 184 were present in the panel samples at different ratios to the wild type. The presence of mutant genotypes was determined qualitatively and quantitatively. All laboratories reported the presence of sequence heterogeneities at codons 41, 215, and 184 in one or more of the panel samples, though not all reported the correct codon genotypes. Two laboratories reported a mutant genotype in samples containing only the wild type, whereas two and three laboratories failed to detect the mutant genotypes at codons 41 and 215, respectively, in a completely mutant DNA population. Mutations present at relative concentrations of 25% of the total DNA population were successfully identified by 13 of 23, 10 of 23, and 16 of 23 labs for codons 41, 215, and 184Val, respectively. For more than 80% of those laboratories that qualitatively detected the presence of a mutation correctly, the estimated wild type/mutant ratio was less than 25% different from the input ratio in those samples containing 25 to 50% or 75% mutant input. This first multicenter study on the quality of DNA sequencing approaches for identifying HIV-1 drug resistance mutations revealed large interlaboratory differences in the quality of the results. The application of these procedures in their current state would in several cases lead to inaccurate or even incorrect diagnostic results. Therefore, proper quality control and standardization are urgently needed.

Effect of weekly adefovir (PMEA) infusions on HIV-1 virus load: results of a phase I/II study.

The compound 9-(2-phosphonylmethoxyethyl)adenine (adefovir; PMEA) is a potent inhibitor of a number of viruses in vitro, such as human immunodeficiency virus (HIV) type 1 and 2, herpes simplex virus (HSV) type 1 and 2, human papillomavirus virus (HBV) and Epstein-Barr virus (EBV). Adefovir also proved to be effective in vivo against feline immunodeficiency virus (FIV) in cats and simian immunodeficiency virus (SIV) in rhesus monkeys. In an open, non-placebo-controlled trial the antiviral activity of weekly doses of adefovir in nine patients with AIDS or AIDS-related complex was studied for a period of 11 weeks. CD4 cell counts at baseline were between 10 and 450 cells/mm3, HIV-1 RNA levels at baseline were between 24,210 copies/ml and 406,197 copies/ml. The drug was administered intravenously at a dose of 1000 mg every week and plasma viral load was assessed at multiple points during the study. Administration of adefovir was tolerated well and no severe side effects were seen. The response to adefovir treatment differed widely between patients. The increase in CD4 cell count at end point ranged from -40 to 120 cell/mm3. The lowest HIV RNA levels were measured after 3-5 days, showing an increase thereafter. The nadir in viral load was achieved after 2 weeks, with a mean viral load decline of 0.7 from baseline. The decrease of the HIV RNA level at end point ranged from -0.3 log10 to 1.8 log10 with a mean decrease of 0.4 log10. Our results indicate that adefovir given intravenously once weekly has a short-lasting initial antiviral effect. The effect of more frequent dosing requires further evaluation. If adefovir is to be useful clinically, it needs to be combined with other antiviral agents.

Efficient inhibition of both syncytium-inducing and non-syncytium-inducing wild-type HIV-1 by lamivudine in vivo.

OBJECTIVE: To determine the effect of lamivudine (3TC) on syncytium-inducing (SI) and non-SI (NSI) HIV-1 populations in vivo. DESIGN: Responses in virus load and 3TC resistance in 40 3TC-treated subjects were analysed in relation to the presence or absence of SI HIV-1 variants. METHODS: Peripheral blood samples were collected at regular intervals from 40 HIV-1-infected subjects during 3TC treatment. Virus isolates obtained at the start of treatment were typed for SI-capacity by coculture with MT-2 cells. Changes in levels of viral RNA in plasma were determined by quantitative reverse transcriptase PCR. The relative amount of wild-type and mutant virus at codon 184, associated with HIV resistance to 3TC, was determined using a primer-guided nucleotide incorporation assay after amplification of part of the reverse transcriptase gene. In five subjects the frequency of productively infected CD4+ cells with SI or NSI variants was determined in relation to codon 184 genotype. RESULTS: Twenty-six subjects harboured only NSI variants at baseline, whereas 14 subjects also harboured SI variants. Although baseline plasma viral RNA load and CD4 cell counts were different between the two groups, no differences in the response to 3TC therapy were observed for these parameters. In-depth analysis of five subjects showed that the kinetics of virus load changes and emergence of 3TC resistance mutations were similar in plasma and cells, and comparable for the SI and NSI populations present in one individual. CONCLUSIONS: These data show that, in contrast to didanosine and zidovudine, the pressure exerted by 3TC is similar for SI and NSI M184 populations.

Lamivudine-resistant human immunodeficiency virus type 1 variants (184V) require multiple amino acid changes to become co-resistant to zidovudine in vivo.

Exposure of human immunodeficiency virus to the nucleoside analogue lamivudine (3TC) rapidly selects for resistant variants with a valine at codon 184 (M184V) in the catalytic site of reverse transcriptase. In vitro, 184V demonstrated increased enzyme fidelity and suppressed zidovudine resistance. Clinical trials demonstrated that 3TC-zidovudine combination therapy results in a strong and sustained antiviral response. To investigate the role of 184V on in vivo virus evolution, the effect of zidovudine addition in 3TC-pretreated patients harboring 184V was studied. In vivo, no significant change in fidelity was observed with 184V, shown by generation of the classical pattern of zidovudine mutations. Of interest, in contrast to zidovudine monotherapy, in which just one substitution is sufficient for in vivo development of significant zidovudine resistance, multiple substitutions are required for the same level of zidovudine resistance in strains harboring 184V. This need for multiple substitutions may be one of the mechanisms explaining the sustained antiretroviral response of the 3TC-zidovudine combination.

Evaluation of carbon-11-labelled phenoxy bridged fatty acids for studying myocardial fatty acid metabolism with PET.

A phenylene moiety in the chain of fatty acids was expected to impair metabolic degradation. Three phenoxy-containing [11C]carboxyl-labelled fatty acids were synthesized and evaluated in mice and an in vivo tissue distribution study. Of these three, 1[11C]-3-(p-dodecyloxyphenyl)propionic acid (C12C3) showed the most favourable uptake in the myocardium: 1.2% of the injected dose at 30 min p.i., vs. 0.6% for [11C]palmitate. The metabolic stability of C12C3 and [11C]palmitate was assessed by determining the amount of exhaled [11C]CO2 during a 30-min interval after injection. It was found that the phenoxy moiety in the gamma-position did not prevent the metabolic degradation of C12C3: After 30 min 20.7% of the injected dose was exhaled as [11C]CO2 vs. 12.7% for [11C]palmitate.