Design and validation of a low density array (Nosochip) for the detection and identification of the main pathogenic bacteria and fungi responsible for nosocomial pneumonia.

The aim of this study was to be able to amplify and to detect on one array 27 different etiologic agents found in nosocomial pneumonia, some being phylogenetically closely related and others very distant. The assay is based on the use of consensus primers combined with the identification of the resulting amplicons by hybridization on specific capture probes present on an array. Three genes were necessary in order to cover the different pathogens. We took a redundancy of at least two positive spots to confirm the identity of each species. Each probe was present in triplicate on the array. The detection limit was between 10 and 1,000 DNA copies in the assay depending on the bacteria and the probe. The assay was also specific when tested both on reference collection strains corresponding to the 27 species of interest and on 57 other bacterial species of the normal human flora. Accuracy of the assay was assessed on 200 clinical isolates and some polymorphisms were indeed observed for 5 species. Effectiveness of the assay was preliminarily validated on 25 endotracheal aspirates and sputum samples, and the results were in accordance either with the cell culture or with the sequencing. Polybacterial infections were well detected in three samples. The results show that a combination of appropriate polymerase chain reaction (PCR) and redundancy of signals on the array allows specific screening of bacteria belonging to different species and genus and even fungi. The results open the way for a possible molecular detection of bacteria in the clinical diagnostic setting.

Evidence of cardiac myolysis in severe nonischemic heart failure and the potential role of increased wall strain.

BACKGROUND: Myocyte death could play a role in heart failure (HF) irrespective of the presence of coronary artery disease. The study aimed to assess this hypothesis by use of the cardiac troponin I (cTnI) assay. METHODS AND RESULTS: Seventy-one patients with nonischemic HF, New York Heart Association (NYHA) class II-IV, with a normal coronary angiogram and after exclusion of myocardiopathies were evaluated in the study. The control group included 9 healthy subjects and 15 patients hospitalized for severe noncardiac dyspnea. Cardiac TnI concentrations were determined at admission with a research reagent (cTnIus) characterized by a detection limit of 0.026 ng/mL and a high analytic sensitivity of 0.002 ng/mL. cTnIus levels were more than 0.026 ng/mL in 19 HF patients, ranging between 0.027 and 0.463 ng/mL, whereas no cTnIus level was detectable in the control group. With use of a reference assay, only 2 HF patients had abnormal cTnI values. Severe HF was observed in 17 of these 19 patients, assessed by NYHA class IV or by the presence of pulmonary edema. Patients with an increased cTnIus level had a more restrictive mitral Doppler pattern (P <.001) and a more distinctive left ventricular (LV) concentric remodeling (P <.0001), whereas LV ejection fraction was similar in both HF groups. The increased cTnIus level was also associated with a LV wall strain biologic marker (ie, an increased brain natriuretic peptide plasma level) (P <.001). CONCLUSIONS: cTnI assay is a promising biochemical method for detecting cardiac myolysis in HF, independent of the presence of coronary artery disease. This subtle myolysis could be in part related to the severely increased LV wall strain.

Fluid therapy directly interferes with immunoassay for cardiac troponin I.

OBJECTIVE: To investigate interference in cardiac troponin I (cTNI) immunoassay induced by some widely used loading fluids. SETTING: A biochemistry unit of a university hospital. MEASUREMENTS AND RESULTS: Human serum with a cTNI concentration of 0.32 microg/l was diluted at 10, 20, 40, and 80% with saline serum (SS), Plasmion (P) (a modified fluid gelatin), hydroxyethyl starch (HES), and 20% human albumin (Alb). Serum with a cTNI concentration of 1.29 microg/l was diluted at 20, 40, 60, and 80%. Four samples with increasing cTNI concentrations (from 0 to 8.14 microg/l) were diluted at 80% with the four fluids. Differences (delta-C) between expected concentrations resulting from the dilutional effect and those measured with the Access cTNI immunoassay were expressed in microg/l. Statistical analysis was performed using a nonparametric test. No false positivity was observed. At a low cTNI concentration (0.32 microg/l), interference was observed with SS and HES at 40 and 80% dilution and with P and Alb at 80%. When cTNI was 1.29 microg/l, interference was observed with each fluid at a dilution of 20% and increased with the increase in dilution. The highest interference was observed with HES, the lowest with P. When the dilution was at 80% for increasing concentrations of cTNI, the higher the initial cTNI was the higher the interference appeared, mostly with SS and HES. CONCLUSIONS: SS, P, Alb, and HES interfere in this cTNI immunoassay. This interference is higher with SS and HES and is higher when the percentage of hemodilution or real cTNI concentration increases.

Elevated circulating cardiac troponin I in patients with cirrhosis.

It has been shown that certain patients with cirrhosis have asymptomatic cardiac abnormalities that have not yet been explained. Thus, cardiac troponin I, a specific marker of myocardial injury, has been measured in patients with cirrhosis without previous cardiac disease. Thirty-two consecutive patients (age 49 +/- 11) with cirrhosis and normal ECG were selected, 22 of which were alcoholic. Hemodynamic investigations were performed. Left ventricular function and mass were evaluated by echocardiography. Serum creatine kinase MB mass, myoglobin, and cardiac troponin I concentrations were measured. Cardiac troponin I concentrations were elevated in 10 patients (32%) (range 0.06-0.25 microg/L) whereas creatine kinase MB mass and myoglobin were normal in all patients. Abnormal troponin I values were not related to the severity of cirrhosis, to the degree of portal hypertension, or to other hemodynamic values. In contrast, elevated serum cardiac troponin I concentrations were related to a decreased stroke-volume index (P <. 05) and a decreased left ventricular mass (P <.05). These results show a high prevalence of slightly elevated serum cardiac troponin I in patients with cirrhosis, especially in those with alcoholic cirrhosis. Elevated troponin I is associated with subclinical left ventricular myocardial damage. These findings may be linked to a lack of left ventricular adaptation in certain patients with cirrhosis and alcoholic cardiomyopathy.

[Brain natriuretic peptide (BNP) in coronary insufficiency: relationship with left ventricular filling and exercise tolerance]. / Brain natriuretic peptide (BNP) dans l'insuffisance cardiaque: relations avec le remplissage ventriculaire gauche et la tolérance à l'effort.

Brain natriuretic peptide (BNP) is a recently discovered peptide, secreted by the atria and ventricles in response to parietal distension. It was recently proposed as a screening test for left ventricular failure. The authors assayed this peptide at rest in 37 patients with chronic heart failure due to left ventricular systolic dysfunction and another 20 patients with various diseases (respiratory failure, cirrhosis, heart transplantation, "diastolic" heart failure) but normal left ventricular systolic function. A significant increase compared to normal values was observed not only in the group of heart failure patients, but also in patients with all other diseases. BNP was significantly higher in NYHA class IV patients. The relationship between plasma BNP levels and ejection fraction was not significant. On the other hand, a good correlation was observed between BNP and left ventricular filling parameters evaluated by cardiac Doppler: E wave deceleration time (r = -0.53, p = 0.001), E/A ratio: r = 0.57 p = 0.005) or VO2 max (r = -0.55, p < 0.005).

Auxiliary liver transplantation: how to improve regeneration of the native liver by surgery.

The technical factors which could influence regeneration of the native liver (NL) in auxiliary liver transplantation (ALT) for fulminant hepatic failure (FHF) are not well known. We studied NL regeneration according to the location of graft anastomosis in the recipient's portal system (superior mesenteric vein versus portal vein), and graft weight (50% reduced-size versus full-size graft) in a rat model of ALT with 80% reduction of the NL, and graft arterialization. NL regeneration was significantly more obvious when the graft was anastomosed on the recipient's superior mesenteric vein, thus establishing venous flow to the NL from the pancreas, the spleen, and the stomach, and when a full-size graft was used. The influence of portal venous flow on NL regeneration, assessed by 3H[-thymidine incorporation, was measurable as early as day 2. Both technical variables in combination resulted in significantly greater regeneration (ratio weight of NL/body weight at day 30: 2.32 +/- 0.68% versus 1.21 +/- 0.63% respectively, P = 0.02). Early preservation of portal flow to the NL is advisable to maximize NL regeneration in ALT. In any case, this regeneration is not impeded by the use of large auxiliary grafts.

[Prognostic significance of early raised cardiac troponine I in unstable angina. Contribution to the identification of a high-risk sub-group]. / Signification pronostique de l'élévation précoce de la troponine I cardiaque chez les patients en angor instable. Contribution à l'identification d'un sous-groupe à haut-risque.

The authors compared the clinical and angiographic characteristics of 44 patients with unstable angina according to cardiac Troponine I concentrations (TnIc) during early blood sampling and then tried to determine a threshold value to predic the occurrence of cardiac events during the hospital period and after 12 months. Tnlc, creatinine-kinase (CK), CK-MB activity and CK-MB mass were sampled over 48 hours. Forty-five per cent of patients had TnIc > or = 0.1 microgram/L; CK-MB activity and CK-MB mass were detected in 16 and 32% of patients. Age, gender, classification and recurrence of angina, previous cardiac history, risk factors, coronary angiographic appearances were comparable in patients with and without raised TnIc. No major cardiac events occurred during the hospital period in either group. The number of angioplasties and coronary bypass procedures was also comparable. At one year, the incidence of myocardial infarction (N = 4) and death (N = 5) was significantly different in patients with raised Tnlc (33% versus 0% in patients without increased TnIc). However, betablocker therapy was less prescribed in the group with the poorest outcomes and left ventricular dysfunction was also significantly more common in this group. Early elevation of Tnlc could contribute to the identification of a high risk subgroup of patients with unstable angina.

Determination of tryptophan and its kynurenine pathway metabolites in human serum by high-performance liquid chromatography with simultaneous ultraviolet and fluorimetric detection.

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of tryptophan and four metabolites of the kynurenine pathway (kynurenine, 3-hydroxykynurenine, kynurenic acid and 3-hydroxyanthranilic acid) in human serum is described. This new method, which uses both isocratic elution and two on-line connected programmable ultraviolet and spectrofluorimetric detectors, allows the determination of these metabolites, in the physiological ranges, with satisfying specificity and sensitivity within 30 min.

[Beta-2 microglobulin in cerebrospinal fluid in neurology]. / La beta 2-microglobuline du liquide céphalorachidien en neurologie.

OBJECTIVES: beta 2-microglobulin (beta 2-m) is a small molecular weight protein (11 800 Daltons) which can transudate into the cerebrospinal fluid (CSF) in the same manner than albumin. Intrathecal synthesis is a sign of local immuno-stimulation and is correlated with immunoglobulin G. The aim of this study was to ascertain the relationship between beta 2-microglobulin levels in the CSF and neurological diseases. METHODS: beta 2-microglobulin was assayed in the CSF and blood using an immunoenzyme method in 64 patients with multiple sclerosis (n = 14), human immunodeficiency virus (HIV) infection (n = 5), meningitis (n = 12) or a peripheral neurological disease (n = 10) and in 7 control subjects. RESULTS: There was no overall correlation between beta 2-m in the CSF and blood levels (r = 0.35). In controls, beta 2-m CSF and blood levels were respectively 0.94 +/- 0.22 and 1.46 +/- 0.83 mg/l. beta 2-m was significantly higher in the CSF of patients with meningitis and in the HIV positive patients (4 +/- 3.5 and 3.69 +/- 2.06 mg/l respectively) (p < 0.05). Type of meningitis (bacterial or non-bacterial) had no effect on the CSF level. For the HIV patients, the CSF/blood ratio for beta 2-m was similar to that in controls due to a rise in both blood and CSF. Finally, in patients with multiple sclerosis, there was no significant change in CSF level of beta 2-m. CONCLUSION: beta 2-microglobulin in cerebrospinal fluid was not found to be correlated with the neurological diseases studied and cannot be used as a diagnostic test.

Comparison of jet and ultrasonic nebulizers for alveolar targeting of methylprednisolone.

The secondary systemic effects of oral corticosteroid therapy in chronic lung disease indicate the possible benefits of local therapy. The aim of this study was to show if alveolar targeting of a corticosteroid, methylprednisolone (MP), is possible, and to determine which type of nebulizer allows the most selective deposition into the alveoli. A jet nebulizer (Respirgard II) with 2 ml volume fill (R2), and an ultrasonic nebulizer (Ultraneb 99) with 4 ml volume fill (U4), were compared using a 40-mg dose of MP labelled 99Tcm human serum albumin. Particle size and MP-to-albumin binding were measured in the aerosol cloud. Each nebulizer was used in random order in five healthy volunteers. A dynamic posterior scan of 68 images of 15 s each was performed with a Gammatome II gamma camera during inhalation. Peripheral and central regions of interest were automatically defined with reproducible methods, and the peripheral-to-central ratio was used as a penetration index. Stomach and oropharynx activities were estimated on static anterior and static left lateral views, respectively, at the end of the examination. The mass median aerodynamic diameter (MMAD) was lower for R2 when unlabelled MP was used. The MMAD of MP+HSA was compatible with alveolar targeting. In the aerosol cloud, MP-albumin binding was 75% for R2 and 79% for U4. Peripheral and central activities at equilibrium (13-16 min) were higher with U4, but the penetration index was significantly higher with R2. Moreover, the stomach and oropharynx activities were significantly lower with R2.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of erythrocyte transketolase activity with thiamine and thiamine phosphate ester levels in chronic alcoholic patients.

The effect of chronic alcoholism on biochemical evaluation of thiamine status was studied by the concomitant determination of erythrocyte transketolase (ETK) activity, its relative increase by in vitro addition of thiamine diphosphate (TDP effect) and the direct measurement of thiamine and its phosphate esters by high performance liquid chromatography. Thirty-eight percent of alcoholic subjects showed a thiamine deficiency with decreased thiamine diphosphate concentrations compared with healthy subjects (90.8 +/- 25.7 nmol/l vs. 176 +/- 28.0 nmol/l, respectively, mean +/- S.D., P < 0.001). Thiamine diphosphate concentrations were highly correlated with total thiamine concentrations and TDP effect (respectively r = 0.99 and 0.79, n = 85, P < 0.001). No abnormality in thiamine phosphorylation related to chronic alcoholism was noted. Finally, 47% of these deficient alcoholic patients had normal ETK activity. We concluded that, if indirect evaluation of thiamine status is to be chosen, the determination of ETK activity should be associated with TDP effect since the latter has been shown to be highly linked to total thiamine and thiamine diphosphate in erythrocytes. Furthermore, the direct measurement of thiamine and its phosphate esters was a more sensitive and specific index of thiamine nutrition.

Determination of thiamine and its phosphate esters in human erythrocytes by high-performance liquid chromatography with isocratic elution.

A high-performance liquid chromatographic method for the simultaneous determination of thiamine and its phosphate esters in human erythrocytes, using postcolumn derivatization, is presented. The sample preparation and the choice of the analytical column avoid the use of an elution gradient. The four thiamine compounds (thiamine and thiamine monophosphate, diphosphate and triphosphate) are eluted within less than 15 min with a detection limit of ca. 20 fmol. The reproducibility and accuracy of the assay are satisfactory. Normal physiological red blood cell concentrations of the four thiamine compounds are included.

Presence of Apo B48, and relative Apo CII deficiency and Apo CIII enrichment in uremic very-low density lipoproteins.

In the present study, lipid and apolipoprotein composition of very low density lipoprotein (VLDL) was analyzed in 39 patients with end-stage renal failure by comparison with 41 healthy subjects. Uremic patients had an increase of serum triglycerides (TG) concentration by comparison with control values. This increase of serum TG was associated with an increase of VLDL which had a normal percent amount of main components. Furthermore a mid-band between VLDL and low density lipoproteins (LDL) on polyacrylamide gel was observed in 22 out of 39 uremic patients but in only 1 out of 41 control subjects. In uremic VLDL Apo B48 was more frequently observed than in control VLDL (p less than 0.05). Furthermore, the content of Apo CII expressed as percent of total Apo C was significantly (p less than 0.001) decreased in uremic VLDL (19.13 +/- 4.54 p. cent) as compared to normal VLDL (23.57 +/- 4.40 p. cent). Apo CIII-O was significantly (p less than 0.001) increased (9.58 +/- 7.19 p. cent vs 5.55 +/- 6.12 p. cent, whereas Apo CIII-1 and Apo CIII-2 distribution was not modified in uremic VLDL. These anomalies were present in uremic patients even when no elevation of fasting serum TG was present. No significant change was observed in uremic patients before their fifth as compared to their first hemodialysis (HD) session, respectively, for any of the parameters studied. Advanced chronic renal failure is associated with a variety of anomalies of TG-rich lipoproteins isolated at d less than 1.006 g/ml which are not reflected by the degree of hypertriglyceridemia and are not corrected by the first four HD sessions.

Influence of haemodialysis membranes on beta 2-microglobulin kinetics: in vivo and in vitro studies.

Amyloidosis of the beta 2-microglobulin (beta 2M) type is a recently recognised complication of dialysis. We studied plasma and ultrafiltrate beta 2M in 36 chronic haemodialysis patients. Long-term dialysis with standard cuprophan membrane in non-oliguric patients and with the AN69 polyacrylonitrile membrane in oliguric patients resulted in lower plasma beta 2M concentrations (means +/- SD, 24.4 +/- 6.6 and 33.0 +/- 8.2 micrograms/ml, respectively) than with cuprophan in oliguric patients (47.0 +/- 13.2 micrograms/ml, P less than 0.01). In acute studies, plasma beta 2M (corrected for haemo-concentration) increased, although not significantly, during cuprophan dialysis (n = 10) from 40.6 +/- 12.2 to 44.8 +/- 7.6 micrograms/ml and decreased during AN69 dialysis (n = 10) from 39.4 +/- 18.0 to 24.3 +/- 7.1 micrograms/ml (P less than 0.02). After 15 min ultrafiltration, beta 2M sieving coefficient in vivo was 0.33 for AN69 but near zero for cuprophan. Total mass transfer of beta 2M across the AN69 membrane during a 4-h dialysis was 142 +/- 45.8 mg (n = 7). AN69, but not cuprophan, membrane fragments incubated in vitro with normal or uraemic plasma exhibited avid [125-I] beta 2M binding (respectively 12.9% vs 0.47% and 6.09% vs 0.06% of total beta 2M bound at 30 min, n = 6, P less than 0.001 for both). No in vitro generation of beta 2M from whole normal or uraemic blood could be demonstrated during incubation with either membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum lipoprotein disturbances in primary and secondary hyperparathyroidism and effects of parathyroidectomy.

Serum lipoprotein disturbances were studied in 86 patients with primary (I), and 34 hemodialysis patients with severe secondary (II), hyperparathyroidism (HPTH) before and seven to 14 days after parathyroidectomy (PTx). In addition, a subset of patients had repeat studies more than 12 months after PTx. In patients with I as well as with II HPTH, mean +/- SEM fasting serum concentrations of total triglycerides (TG) (1.51 +/- 0.09 and 2.17 +/- 0.19 mmol/L, respectively) were significantly increased when compared with that of 22 age- and sex-matched healthy control subjects (1.01 +/- 0.09 mmol/L, P less than .001). No consistent anomalies of serum total cholesterol and lipoprotein cholesterol content were observed in Io HPTH patients. In uremic II HPTH patients, the cholesterol content of high-density lipoprotein (HDL) was significantly (P less than .01) depressed, compared with normal subjects. In the short term, PTx normalized serum total TGs (P less than .001) in Io HPTH patients from 1.50 +/- 0.11 to 1.19 +/- 0.07 mmol/L seven days after PTx. The surgical correction of II HPTH in dialysis patients was also followed by an improvement of hypertriglyceridemia from 2.22 +/- 0.21 to 1.46 +/- 0.08 mmol/L and 1.46 +/- 0.09 mmol/L seven and 14 days, respectively, after PTx (P less than .01). Long-term follow-up after PTx shows clearly a persistent decrease in serum TG concentration in I HPTH patients (1.17 +/- 0.11 mmol/L), as well as in II HPTH patients (1.61 +/- 0.18 mmol/L), 12 months after PTx by comparison with values determined before PTx.(ABSTRACT TRUNCATED AT 250 WORDS)