Specific real-time PCR for simultaneous detection and identification of Legionella pneumophila serogroup 1 in water and clinical samples.

Legionella pneumophila, a bacterium that replicates within aquatic amoebae and persists in the environment as a free-living microbe, is the causative agent of Legionnaires' disease. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease, and within the 15 serogroups (Sg), L. pneumophila Sg1 causes more than 84% of Legionnaires' disease worldwide. Thus, rapid and specific identification of L. pneumophila Sg1 is of the utmost importance for evaluation of the contamination of collective water systems and the risk posed. Previously we had shown that about 20 kb of the 33-kb locus carrying the genes coding for the proteins involved in lipopolysaccharide biosynthesis (LPS gene cluster) by L. pneumophila was highly specific for Sg1 strains and that three genes (lpp0831, wzm, and wzt) may serve as genetic markers. Here we report the sequencing and comparative analyses of this specific region of the LPS gene cluster in L. pneumophila Sg6, -10, -12, -13, and -14. Indeed, the wzm and wzt genes were present only in the Sg1 LPS gene cluster, which showed a very specific gene content with respect to the other five serogroups investigated. Based on this observation, we designed primers and developed a classical and a real-time PCR method for the detection and simultaneous identification of L. pneumophila Sg1 in clinical and environmental isolates. Evaluation of the selected primers with 454 Legionella and 38 non-Legionella strains demonstrated 100% specificity. Sensitivity, specificity, and predictive values were further evaluated with 209 DNA extracts from water samples of hospital water supply systems and with 96 respiratory specimens. The results showed that the newly developed quantitative Sg1-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk L. pneumophila Sg1 in water and clinical samples.

Legionella pneumophila - a human pathogen that co-evolved with fresh water protozoa.

The bacterial pathogen Legionella pneumophila is found ubiquitously in fresh water environments where it replicates within protozoan hosts. When inhaled by humans it can replicate within alveolar macrophages and cause a severe pneumonia, Legionnaires disease. Yet much needs to be learned regarding the mechanisms that allow Legionella to modulate host functions to its advantage and the regulatory network governing its intracellular life cycle. The establishment and publication of the complete genome sequences of three clinical L. pneumophila isolates paved the way for major breakthroughs in understanding the biology of L. pneumophila. Based on sequence analysis many new putative virulence factors have been identified foremost among them eukaryotic-like proteins that may be implicated in many different steps of the Legionella life cycle. This review summarizes what is currently known about regulation of the Legionella life cycle and gives insight in the Legionella-specific features as deduced from genome analysis.

Interleukin-1 beta-mediated glucose uptake by chondrocytes. Inhibition by cortisol.

The aim of this study was to investigate the in vitro effects of interleukin-1 beta (IL-1 beta) on cultured human articular chondrocytes from patients with osteoarthritis, by the evaluation of glucose uptake. We also investigated the inhibitory effect of cortisol on IL-1 beta-mediated glucose uptake. Experiments were performed by using 2-deoxy-D-[1-3H]glucose (2-DOG) and confluent monolayer cells at first passage. Confluent cells were also treated for 24 h with different concentrations of cortisol (10(-5), 10(-6) and 10(-7) mol/l). IL-1 beta (100 pg/ml) was added 6 h before glucose uptake studies. Glucose uptake stimulation was observed 3 h after the addition of 100 pg/ml IL-1 beta (+70%) and increased up to 24 h (+145%). The sensitivity and responsiveness of chondrocytes to IL-1 beta, studied after a 6 h association time, appeared to be dose-dependent from 0.1 pg/ml IL-1 beta (+50%) to 100 pg/ml (+130%) over basal values. The effect of the cytokine was protein synthesis-dependent, as demonstrated by using cycloheximide. Cortisol inhibited the action of IL-1 beta on glucose uptake because it reduced stimulating effects by 28% at concentrations as weak as 10(-6) mol/l. Results appeared similar when IL-1 beta and cortisol were added simultaneously 6 h before 2-DOG uptake. The rapid effect of cortisol was protein-synthesis dependent, as indicated by inhibition by cycloheximide. These results suggest that IL-1 beta stimulates chondrocyte metabolic activity. The inhibition of IL-1 beta-mediated glucose uptake is suggested for studying the anti-IL-1 effect of other anti-rheumatic drugs.

Preliminary clinical studies of a biological skin equivalent in burned patients.

The possibility of covering large areas of full thickness skin loss with 'living skin equivalent' produced by a modification of Bell's method was studied. Living skin equivalents, composed of a dermal equivalent (fibroblasts plus collagen) covered by epithelial cells were grafted, meshed or non-meshed, onto granulation tissue and, in one patient, onto fascia. Eight patients with full skin thickness burn wounds covering over 15 per cent of the body surface area were thus partially covered. The graft 'take' was evaluated every 48 h. In every patient grafted, an extensive lysis (60-90 per cent) of the skin equivalent graft was observed at the first dressing (48 h). In one patient only, a significant percentage of 'take' (40 per cent) was observed 14 days after grafting. These disappointing results were probably related to the presence of collagenases or proteases produced on the wound bed either by bacteria or by surrounding human cells. It appears that at the present time the biochemical nature of the dermal equivalent used is not yet completely appropriate to serve routinely as a substitute for human skin.

Plasma fibronectin time course in burned patients: influence of sepsis.

Plasma fibronectin time course was studied in 30 patients versus clinical criteria: body surface area burn (BSA), unit burn standard (UBS), and infection state. The mean value for all the population was lowest within days 1-3. No correlation was observed between fibronectin concentration and clinical criteria, either at admission or subsequently. Fibronectin levels of nonseptic patients (Group 1) did not differ significantly during the first days from those of patients who, at any time of their hospitalization, would become moderately infected (Group 2) or septic (Group 3). Afterwards, protein concentration of Group 1 increased regularly and hyperopsonization was observed from day 17 until the end of the study, day 35. In contrast, protein levels of Groups 2 and 3 remained low with a further improvement for moderately infected patients. A marked and significant difference was observed between nonseptic patients and septic patients, from the 6th postburn day. Variation of Groups 2 and 3 may be related to the infectious period, whatever the BSA or UBS index evolution. Since infection was related to low fibronectin level in the three deaths observed, it appears that opsonic protein cryoprecipitate would be of value in the treatment of burn victims.

New statistical approach in biochemical method-comparison studies by using Westlake's procedure, and its application to continuous-flow, centrifugal analysis, and multilayer film analysis techniques.

In this paper, we present an application of Westlake's method (Biometrics 32:741, 1976) in method-comparison studies in clinical chemistry. The techniques under study are continuous flow (CF), centrifugal analysis (CA), and multilayer film analysis (the Kodak Ektachem procedure KE). The experimental data are for plasma calcium and serum uric acid. It results from a particular regression procedure proposed by York (Can J Phys 44:1079, 1966) that the usual regression comparison technique (joint testing procedure) is irrelevant because it rejects the identity hypothesis for the three methods, whereas fixing a tolerable upper limit deviation, delta, between two methods would lead to the conclusion that, in 95% of cases, CF and KE results are equivalent for plasma calcium (delta less than or equal to 45 mumol/L) and CF is roughly equivalent to the other two methods for serum uric acid (delta less than or equal to 27 mumol/L).

[Determination of the prothrombin time and of the activated cephalin time wit semi-automatic Electra 600 apparatus (author's transl)]. / Détermination du temps de Quick et du temps de céphaline activée avec l' appareillage semi-automatique Electra 600.

This work studies the quality of the response of the Electra 600 for the determination of the prothrombin time (PT) and the activated cephalin time (ACT). The intra-serial precision was good for the PT and the ACT with normal or pathological plasmas (CV 2%). The precision was excellent for the PT (r = 0.98; Electra 600-fibrometer). The correlation coefficient varies between 0.85 and 0.95 for the ACT depending on the nature of the activator chosen. Lactescent plasmas having a protein concentration greater than 85 g/l should be treated manually for measurement of the PT. No interference was noted with other biological substances: eg glucose, bilirubin, hemoglobin. The increasing addition of heparin indicates a correct sensitivity of the response with Electra 600 both for short and long ACT. Autoanalysis renders the determinations of PT and ACT independant of the manipulator, that of the PT being much more rapid than with the manual method

[Comparative study of digoxin determination by E.L.I.S.A. and radioimmunoassay techniques (author's transl)]. / Etude comparative du dosage de la Digoxine ar méthodes Immunoenzymologique (E.L.I.S.A.) et radio-immunologique.

This study is based on a double blind interlaboratory comparison between radioimmunoassay and E.L.I.S.A. digoxin determination. The correlation between digoxin values found with these two methods is good (r = 0.96 for therapeutic and toxic ranges 1,0 ng/ml - 5,5 ng/ml). The results indicate good repeatability, reproducibility and precision. The determination by E.L.I.S.A. can be performed with sera or plasma. The presence of haemolysis makes no appreciable difference in results. No variation is observed when different kits are used from an identical lot. However digoxin gives an important cross reactivity with digitoxin in Enzymeimmunoassay. Therefore it is necessary to know exactly the digitalis glycoside before the determination in order to avoid significant error.