Rapid Microbiology Screening in Pharmaceutical Workflows.

Recently advances in miniaturization and automation have been utilized to rapidly decrease the time to result for microbiology testing in the clinic. These advances have been made due to the limitations of conventional culture-based microbiology methods, including agar plate and microbroth dilution, which have long turnaround times and require physicians to treat patients empirically with antibiotics before test results are available. Currently, there exist similar limitations in pharmaceutical sterility and bioburden testing, where the long turnaround times associated with standard microbiology testing drive costly inefficiencies in workflows. These include the time lag associated with sterility screening within drug production lines and the warehousing cost and time delays within supply chains during product testing. Herein, we demonstrate a proof-of-concept combination of a rapid microfluidic assay and an efficient cell filtration process that enables a path toward integrating rapid tests directly into pharmaceutical microbiological screening workflows. We demonstrate separation and detection of Escherichia coli directly captured and analyzed from a mammalian (i.e., CHO) cell culture with a 3.0 h incubation. The demonstration is performed using a membrane filtration module that is compatible with sampling from bioreactors, enabling in-line sampling and process monitoring.

Value of the proximal flow convergence method for quantification of the regurgitant volume in mitral regurgitation Influence of the mechanism of regurgitation, the imaging of the flow convergence region, and different calculation modalities.

UNLABELLED: The purpose of this study was to evaluate whether the underlying mechanism of mitral regurgitation influences the reliability of the proximal flow con- vergence method to assess the regurgitant volume. Furthermore, the mode of imaging the flow convergence region and different correction algorithms for calculation of the regurgitant volume were compared. METHODS: Regurgitant volume was assessed in 45 patients (age 61+/-13 years) with organic (n=19) and functional (n=26) mitral regurgitation by the proximal flow convergence method for aliasing velocities between 14 and 64 cm/s using two-dimensional color Doppler imaging. Different correction and calculation algorithms were compared. In addition, regurgitant volume was determined using color Doppler M-mode for an aliasing velocity of 28 cm/s. The quantitative Doppler method was used as reference. RESULTS: In organic mitral regurgitation correlation coefficients (mean differences) between the proximal flow convergence method and the reference method were 0.25-0.43/ 0.58-0.67 (46-111 ml/15-17 ml) before/after geometric correction of the regurgitant volume for the aliasing velocities investigated. The correlation coefficient (mean difference) using color Doppler M-mode imaging was 0.68 (85 ml). The corresponding values in functional mitral regurgitation were 0.74-0.88/0.74-0.88 (-5-8 ml/-7-5 ml) for two-dimensional color Doppler and 0.88 (-1 ml) for M-mode imaging. CONCLUSIONS: The regurgitant volume was overestimated by the proximal flow convergence method in organic mitral regurgitation irrespective of the application of different correction algorithms or the use of color Doppler M-mode. A sufficiently reliable determination of the regurgitant volume by the proximal flow convergence method was possible in functional mitral regurgitation. In that case a simplified calculation of the regurgitant volume based on the proximal flow convergence method was feasible.

Biological properties of poly-L-lysine-DNA complexes generated by cooperative binding of the polycation.

We have evaluated the effect of NaCl concentration on the mode of binding of poly-L-lysine to DNA and the resulting structural and functional features of the condensed DNA particles using DNA precipitation, DNase I resistance, electron microscopy, and receptor-mediated gene transfer assays. At a high concentration of NaCl and in the presence of excess DNA, poly-L-lysine interacted with DNA cooperatively, fully condensing some of the DNA and leaving the rest of the DNA unbound. At low NaCl concentrations, poly-L-lysine molecules interacted with DNA in a noncooperative fashion, i.e. they bind randomly to the whole population of DNA molecules. Cooperative binding of poly-L-lysine to DNA occurred over a narrow range of NaCl concentrations, and the specific salt concentration depended on the length of the poly-L-lysine. The ability of condensed DNA to withstand digestion by DNase I was correlated with the structural features of the condensed DNA as determined by electron microscopy. Using our condensation procedure, cooperative binding of poly-L-lysine to DNA is a necessary prerequisite for the preparation of condensed DNA having a spherical shape and a diameter of 15-30 nm. Condensed DNA, containing galactosylated poly-L-lysine, was evaluated further for the extent and specificity of receptor-mediated gene transfer into HuH-7 human hepatoma cells via the asialoglycoprotein receptor. Efficient receptor-mediated transfection occurred only when condensed DNA complexes had a spherical shape with a diameter of 15-30 nm; asialofetuin, a natural ligand for the asialoglycoprotein receptor, inhibited this process by up to 90%. Our results support the importance of appropriate DNA condensation for the uptake and ultimate expression of DNA in hepatic cells.

Alpha-galactosidases from the larval midgut of Tenebrio molitor (Coleoptera) and Spodoptera frugiperda (Lepidoptera).

There are three midgut alpha-galactosidases (TG1, TG2, TG3) from Tenebrio molitor larvae that are partially resolved by ion-exchange chromatography. The enzymes have approximately the same pH optimum (5.0), pl value (4.6) and Mr value (46000-49000) as determined by gel filtration or native electrophoresis run in polyacrylamide gels with different concentrations. Substrate specificities and functions were proposed for the major T. molitor midgut alpha-galactosidases (TG2 and TG3) based on chromatographic, carbodiimide inactivation, Tris inhibition, and on substrate competition data. Thus, TG2 would hydrolyse alpha-1,6-galactosaccharides, exemplified by raffinose, whereas TG3 would act on melibiose and apparently also on digalactosyldiglyceride, the most important compound in the thylacoid membranes of chloroplasts. Most galactoside digestion should occur in the lumen of the first two thirds of T. molitor larval midguts, since alpha-galactosidase activity predominates there. Spodoptera frugiperda larvae have three midgut alpha-galactosidases (SG1, SG2, SG3) partially resolved by ion-exchange chromatography. The enzymes have similar pH optimum (5.8), pl value (7.2) and Mr value (46000-52000), and at least the major alpha-galactosidase must have an active carboxyl group in the active site. Based on data similar to those described for T. molitor, SG1 and SG3 should hydrolyse melibiose and SG3 should digest raffinose and, perhaps, also digalactosyldiglyceride. The midgut distribution of alpha-galactosidase activity supports the proposal that alpha-galactosidase digestion occurs at the surface of anterior midgut cells in Spodoptera frugiperda larvae.

[Comparison of efficacy, safety and cost-effectiveness of intravenous versus oral propafenone in paroxysmal atrial fibrillation]. / Porównanie skutecznosci, bezpieczenstwa i kosztów leczenia dozylna i doustna postacia propafenonu u chorych z napadowym migotaniem przedsionków.

The purpose of this study was to investigate the efficacy, safety and cost-effectiveness of intravenous and oral propafenone in the conversion of paroxysmal atrial fibrillation propafenone to sinus rhythm. We analysed two groups of 100 consecutive patients (pts) treated because of propafenone with duration < 48 h. The first group was treated with intravenous PFN (bolus of 70 to 140 mg) and the second group was treated with oral PFN (300 to 600 mg). These 2 groups were comparable in age, sex, evidence of CAD, hypertension, mitral valve disease, history of hyperthyroidism and the level of K+ at admittance. Conversion to sinus rhythm was achieved in 64 (64%) pts who received i.v. propafenone and in 77 (77%) who received oral propafenone (p < 0.05). We conclude that oral and intravenous propafenone is safe in the termination of propafenone. Oral route of administration appears to be superior to intravenous because of greater efficacy and cost-effectiveness.

Influence of the orifice inlet angle on the velocity profile across a flow convergence region by color Doppler in vitro.

The converging flow field proximal to a leaking valve is determined among other things by the orifice inlet angle formed by the leaflets. Thus, the inlet angle affects the determination of regurgitant flow rate by the flow convergence method. Based on the hypothesis of spheric isovelocity surfaces, others had postulated that a local velocity within the flow convergence should change inversely proportional to changes in the three-dimensional inlet angle. This concept would allow correction of the determination of regurgitant flow for nonplanar orifice inlet angles. We tested this concept in vitro. In a flow model, the flow convergence region proximal to different orifice plates was imaged by color Doppler: funnel-shaped, planar and tip-shaped (inverted funnels) orifice plates, with circular orifices of 2- and 7-mm diameter. Velocity profiles across the flow convergence along the flow centerline were read from the color maps. As predicted, the local velocities were inversely related to the inlet angle, but only at the 2-mm funnel orifices, this effect was inversely proportional to the three-dimensional inlet angle (i.e., in agreement with the mentioned concept). However, for any 7-mm orifice and/or inlet angle of > 180 degrees, the effect of the inlet angle was considerably less than predicted by the aforementioned concept. With increasing orifice diameter and with decreasing distance to the orifice, the effect of the orifice inlet angle was reduced. The effect of the orifice inlet angle on the flow convergence region is modulated by orifice size and the distance to the orifice. Therefore, correction of flow estimates in proportion to the three-dimensional inlet angle will lead to considerable errors in most situations of clinical relevance, namely to massive overcorrection when analyzing velocities located close to wide orifices.

Experimental and theoretical 31P and 77Se nuclear magnetic shielding tensors for bis(dineopentoxyphosphorothioyl) diselenide.

An intergrown crystal of two phases of bis(dineopentoxyphosphorothioyl) diselenide 1 was investigated by goniometer 31P NMR. From the angular dependence of the chemical shift, the tensors of a triclinic and a monoclinic phase were determined. The principal values sigma11, sigma22, and sigma33, of the absolute nuclear magnetic shielding tensors for the triclinic phase are 134.1, 227.2, and 375.5 ppm and for the monoclinic phase are 132.4, 227.8, and 374.2 ppm, respectively. In both cases, the principal axis 3 of the 31P tensor is directed nearly along the P=S bond and the principal axis 2 is nearly perpendicular to the S=P-Se plane. Calculations of the 31P and 77Se nuclear magnetic shielding tensors were performed for molecules of both phases of 1 and for model compounds by the sum-over-states density functional perturbation theory IGLO method. The rms distances between calculated and experimental 31P NMR icosahedral tensor values sigma(j) (j = 1, ..., 6) amount to 17-21 ppm. The calculated and experimental orientations of the 31P principal axes show a maximum difference of 5 degrees and rms distances of 3.2 and 3.3 degrees. For the principal value sigma33 of the selenium shielding tensor the agreement between calculated and experimental values is satisfactory, but the calculated values sigma11 and sigma22 are distinctly too small. Calculations for a model compound in which the methyl groups of the neopentoxy residue are substituted by protons lead practically to the same results.

Experimental neonatal respiratory failure induced by lysophosphatidylcholine: effect of surfactant treatment.

The purpose of this study was to characterize the toxic effects of lysophosphatidylcholine (lyso-PC) on neonatal lung function. Various doses of lyso-PC (from 0 to 40 mg/kg) were administered to near-term newborn rabbits. Lung-thorax compliance during mechanical ventilation was significantly decreased by doses >/=10 mg/kg, and static lung volumes during deflation were decreased by doses >/=20 mg/kg. Using the same experimental model, we investigated the effects of modified porcine surfactant (Curosurf, 200 mg/kg). Animals exposed to lyso-PC at birth and treated simultaneously with surfactant showed a satisfactory therapeutic response, whereas those treated after 30 min failed to respond. These animals also had a much larger leak of albumin into the air spaces and an elevated minimum surface tension of the lavage fluid in a pulsating bubble surfactometer, suggesting inactivation of the exogenous surfactant. Timing of surfactant administration may thus be essential for the therapeutic effect in this experimental model of acute lung injury.

Aortic regurgitant flow by color Doppler measurement of the local velocity 7 mm above the leak orifice--Part 2: Comparison with cardiac catheterization.

AIMS: An in vitro study of the flow convergence region in aortic regurgitation has shown that regurgitant flow rate can be derived from the local velocity V(7 mm) at 7 mm distance above the leak orifice. This clinical study was performed to test this method in patients. METHODS AND RESULTS: In 67 patients with aortic regurgitation, the flow convergence region was imaged by color Doppler. By analogy with the afore mentioned in vitro study, velocity profiles of the acceleration across the flow convergence region were read from the color maps. The profiles were fitted by using a multiplicative regression model. The V(7 mm) was read from the regression curve, and instantaneous regurgitant flow Q was derived from the V(7 mm) with the equation developed in vitro (Q = V(7 mm).cm2/0.28). Q showed a close association with the angiographic grade. Q-derived regurgitant stroke volume correlated significantly with invasive measurements by the angio-Fick method (r = 0.897, SEE = 19.9 ml, y = 0.88x + 5.9 ml). CONCLUSIONS: Within the color Doppler flow convergence region of aortic regurgitation, the local velocity at 7 mm distance to the leak reflects regurgitant flow rate.

Heart-rate variability for discrimination of different types of neuropathy in patients with insulin-dependent diabetes mellitus.

It has been shown that patients with insulin-dependent diabetes mellitus (IDDM) may reveal abnormal alterations in heart-rate variability (HRV) due to autonomic neuropathy. This study was performed to prove whether heart-rate variability can be used to stratify diabetic patients with different types of neuropathy. 48 patients with IDDM (age 17-64 yr) underwent standard function tests to assess autonomic and peripheral neuropathy. According to the results of these tests they were divided into 4 groups: Group 1: 18 patients without autonomic or peripheral neuropathy. Group 2: 13 patients with peripheral neuropathy. Group 3: 7 patients with autonomic neuropathy. Group 4: 9 patients with autonomic and peripheral neuropathy. HRV was measured by continuous 24-hours monitoring and time domain parameters were calculated. The results were compared with sex and age-matched healthy controls according to the individual characteristics of the groups and among each subgroup. Our results showed that in Group 1 there was a significant difference of time domain parameters indicative of parasympathetic influence, i.e. rMSSD and pNN50 in comparison to the control subjects (p = 0.002, p = 0.008). These results depended on the duration of diabetes; a subgroup of patients with a duration of IDDM of less than 2 years had no significant differences of HRV values. Group 2 showed the same significant differences. Group 3 and 4 showed significant differences in all measured time domain variables (SDNN, SDANN, SDNN index, rMSSD and pNN50) in comparison to the control subjects (p < 0.04). A comparison of group 1 with group 2 offered significant differences in rMSSD and pNN50 (p = 0.004, p = 0.003). Comparing group 1 with group 3 and 4, all HRV parameters showed significant differences (p < 0.03). In conclusion, HRV is able to distinguish between patients with different types of neuropathy depending on the involvement of parasympathetic or more sympathetic influenced parameters. Furthermore, this method is able to unmask early manifestations of neurological disorders prior to their detection by neurological function tests.

Comparison of the proximal flow convergence method and the jet area method for the assessment of the severity of tricuspid regurgitation.

AIMS: To compare the value of the proximal flow convergence method and the jet area method for the determination of the severity of tricuspid regurgitation. METHODS AND RESULTS: The proximal isovelocity surface area radius and the jet area/length were measured in 71 consecutive patients with angiographically graded (grade 0/I-III) tricuspid regurgitation. Rank correlation coefficients with the angiographic grade were 0.71 (P < 0.001) for the proximal isovelocity surface area radius (aliasing border of 28 cm.s-1), 0.66 (P < 0.001) for the jet area, and 0.63 (P < 0.001) for the jet length. The proximal isovelocity surface area radius was significantly correlated with the jet area/length (correlation coefficients 0.82/0.77, P < 0.001). Correct differentiation between mild to moderate (grade I-II) and severe (grade III) tricuspid regurgitation was achieved in 62 of 71 patients (87%) by means of the proximal isovelocity surface area radius, in 61 of 71 (86%) by the jet area, and in 62 of 71 (87%) by the jet length. Grade III tricuspid regurgitation was not identified in five of 21 patients (24%) by means of the proximal isovelocity surface area radius, in six of 21 (29%) by the jet area, and in seven of 21 (33%) by the jet length. CONCLUSION: The flow convergence method and the jet area method are of similar value for the determination of the severity of tricuspid regurgitation. Both methods differentiated mild to moderate from severe tricuspid regurgitation in most patients. However, underestimation of severe tricuspid regurgitation in 20-30% of the cases represents a serious limitation of both methods.

Anisotropy of chemical shift and J coupling for P-31 and Se-77 in trimethyl and triphenyl phosphine selenides.

The 31P and 77Se magic angle spinning (MAS) nuclear magnetic resonance (NMR) experiments for selenium-77 enriched (70%) trimethylphosphine selenide 1 and triphenylphosphine selenide 2 were carried out in order to determine the nuclear magnetic shielding tensors of both nuclei and to establish values of the phosphorus-selenium indirect spin-spin coupling anisotropy delta J. The m = +1/2 and m = -1/2 subspectra were analysed by the dipolar-splitting-ratio method of Eichele and Wasylischen. For the C(S) molecule 1, delta J was obtained to be +640 +/- 260 Hz from the 31P spectrum and +550 +/- 140 Hz from the 77Se spectrum. Density functional theory (DFT) calculations give a delta J value of about +705 Hz. The value of delta J could not be determined unambiguously by analysis of the 31P spectra for the C1 molecules 2; nevertheless, an estimation of delta J was possible. The principal axis 3 of the phosphorus shielding tensor was determined to be nearly parallel to the PSe bond in 1 and 2. For the selenium shielding of 1, the same orientation was found, whereas in 2, the principal axis 2 of the selenium shielding was found to be oriented nearly along the PSe bond. The experimentally determined phosphorus nuclear magnetic shielding tensors agree well with those calculated by the IGLO method. For those two principal values of the selenium-shielding tensors corresponding to directions nearly perpendicular to the SeP bond, the agreement between calculated and experimental values is satisfactory. For the third one, corresponding to the principal axis close to the SeP bond, the calculated deshielding contributions are distinctly too small for both compounds investigated. Trends observed for the calculated molecular orbital (MO) contributions to the shielding as well as possible reasons for the underestimation of the deshielding contributions along the SeP bond are discussed.

Quantification of mitral and tricuspid regurgitation by the proximal flow convergence method using two-dimensional colour Doppler and colour Doppler M-mode: influence of the mechanism of regurgitation.

In patients with mitral (n=77: organic=49, functional=28) and tricuspid regurgitation (n=55: functional=54) quantified by angiography, the temporal variation of the proximal flow convergence region throughout systole was assessed by colour Doppler M-Mode, and peak and mean radius of the proximal isovelocity surface area for 28 cm/s blood flow velocity were measured. Additionally, the peak radius derived from two-dimensional colour Doppler was obtained. About 50% of the patients with mitral and tricuspid regurgitation showed a typical temporal variation of the flow convergence region related to the mechanism of regurgitation. The different proximal isovelocity surface area radii were similarly correlated to the angiographic grade in mitral and tricuspid regurgitation (rank correlation coefficients 0.55-0.89) and they differentiated mild to moderate (grade < or =II) from severe (grade > or =III) mitral and tricuspid regurgitation with comparable accuracy (82-96%). However, moderate mitral regurgitation due to leaflet prolapse in two patients was correctly classified by the mean M-mode radius and overestimated by both peak radii. Only half of the patients showed a typical variation of the flow convergence region related to the mechanism of regurgitation. The different proximal isovelocity surface area radii were suitable to quantify mitral and tricuspid regurgitation in most patients. However, in mitral regurgitation due to leaflet prolapse the use of the mean M-mode radius may avoid overestimation.

Pathophysiology of neonatal lung injury induced by monoclonal antibody to surfactant protein B.

Near-term newborn rabbits were exposed via the airways to a monoclonal antibody to surfactant protein B and ventilated for 0-120 min. Control animals received nonspecific rabbit or mouse immunoglobulin G, saline, or no material via the airways. Administration of the antibody at > or = 40 mg/kg elicited an immediate, significant fall in lung-thorax compliance associated with progressive intra-alveolar edema and/or alveolar collapse and necrosis and desquamation of airway epithelium, and hyaline membranes. The vascular-to-alveolar leak of human albumin and human immunoglobulin G, injected intravenously at birth and determined in lung lavage fluid 60-120 min after instillation of the antibody, was 1.8% for the left lung, with no difference between the markers. The average leak in control animals ventilated for 120 min was < 0.3% (P < 0.05). Cytospin preparations of lung lavage fluid from animals exposed to the antibody showed significantly increased recruitment of neutrophilic granulocytes. The pathology and pathophysiology of neonatal lung injury induced by the monoclonal antibody to surfactant protein B probably reflect a combination of direct inactivation of surfactant and an inflammatory response triggered by the immune reaction.

Signal-averaged electrocardiogram in patients with insulin-dependent (type 1) diabetes mellitus with and without diabetic neuropathy.

The purpose of this study was to investigate the presence of ventricular late potentials derived from signal-averaged ECG in patients with IDDM with and without diabetic neuropathy. Eighty patients with IDDM but without evidence of cardiac disease and 80 age-matched healthy control subjects were investigated. The corrected QT interval was measured from the standard surface electrocardiogram. Ventricular late potentials were derived from signal-averaged electrocardiogram. Out of the 80 diabetic patients, 20 had an autonomic neuropathy, 20 had an isolated peripheral neuropathy, and 40 had no symptoms of neuropathy. The corrected QT interval was significantly prolonged in patients with an autonomic neuropathy as compared with the control group (436 +/- 23 ms(x 5) vs 384 +/- 23 ms(x 5), p < 0.001). In the other patient groups there was no significant prolongation of the corrected QT interval. Ventricular late potentials were present in 3 diabetic patients with an isolated peripheral neuropathy and in 1 control subject (NS). No diabetic patient with an autonomic neuropathy had ventricular late potentials. Our data did not indicate an increased incidence of ventricular late potentials derived from signal-averaged electrocardiogram in diabetic patients independent of a coexisting diabetic neuropathy or a prolonged corrected QT interval.

Biochemical and functional characterization of DNA complexes capable of targeting genes to hepatocytes via the asialoglycoprotein receptor.

Electrostatic binding of polycations or basic polypeptides to the DNA phosphate backbone has been previously described as a one-step process which results in uncontrolled aggregation and precipitation of the DNA in solution. We describe here a multistep process in which the condensation of DNA in the presence of poly-L-lysine can be controlled to produce particles of discrete size and shape suitable for receptor-mediated gene transfer in vivo and in vitro. The first step in this process involves the gradual accretion of poly-L-lysine onto the DNA phosphate backbone, until charges are neutralized. The addition of poly-L-lysine to a concentrated solution of DNA in this fashion prevents intermolecular aggregation of the DNA, presumably by promoting the formation of a nucleus of condensation along the length of each DNA molecule. The second stage of the process involves adjusting the ionic strength of the solvent to facilitate the solubilization of compact DNA.poly-L-lysine complexes. Several physical and biochemical parameters have been studied and correlated with the efficacy of DNA/ligand-poly-L-lysine particles in transferring genes to the liver of adult animals by receptor-mediated endocytosis.

Influence of pulse repetition frequency and high pass filter on color Doppler maps of converging flow in vitro.

Assessment of regurgitant flow by the flow convergence method is based on reading absolute velocities from color Doppler maps. Velocity overestimation by high pass filtering above 100 Hz has been reported. An extremely low filter, however, is impracticable in patients. A ratio of pulse repetition frequency (PRF)/filter of 10/1 usually results in good quality color maps as judged visually. We studied in vitro the influence of RPF and filter on the absolute velocities within color maps of the flow convergence, keeping PRF/filter at 10/1. The color maps were also compared with computerized flow simulations. Flow across different orifice plates was scanned using two different setups for each flow condition: low velocity setup (PRF 600-2500 Hz, filter 50-300 Hz) and high (PRF 1500-6000 Hz, filter 200-600 Hz). From the color maps, velocity profile curves were read along the flow center line across the flow convergence. The high velocity setup provided artefact-free color maps at a distance d = 2-4 through 8-11 mm to the orifice, the low setup at d = 6-8 through 18 mm. Within the overlapping range (d = 6-8 through 8-11 mm), the resulting curves showed no significant differences in local velocity, with a slight trend towards higher velocities with the high velocity setup (2.2-2.9%). The simulations agreed well with color Doppler except for slightly lower values at d > 10-12 mm. Changes in PRF and filter have no significant influence on the absolute velocities displayed within color maps as long as PRF/filter is kept close to 10/1.

[Unmasking an exclusively retrograde accessory pathway by catecholamines]. / Demaskierung einer ausschliesslich retrograd leitenden akzessorischen Bahn durch Katecholamine.

A 52-year-old man with frequent episodes of narrow QRS complex tachycardias with rates of 150/min was admitted for electrophysiological evaluation and treatment. P waves could be seen in the ST-segment of the surface ECG during tachycardia. Atrial stimulation during electrophysiological testing was not able to induce tachycardia. During atrial stimulation, there was no evidence of conduction via an accessory pathway or of dual AV node conduction properties. Ventricular stimulation showed complete ventriculoatrial block. After intravenous administration of the catecholamine orciprenaline, single atrial extrastimuli induced an AV macro-reentrant tachycardia with a rate of 165/min. VA conduction showed the earliest retrograde atrial activation in the left anterolateral area. Thus, there was an accessory pathway which only conducted in ventriculoatrial direction and only during adrenergic stimulation. After successful radiofrequency catheter ablation, complete ventriculoatrial block was recorded even after repeat administration of orciprenaline during ventricular stimulation. This case confirms the need to administer catecholamines in every undiagnosed tachycardia during electrophysiological testing to reveal the mechanism of the tachycardia.