Hervé Delacour

Molecular Characterization of blaNDM-1 in a Sequence Type 235 Pseudomonas aeruginosa Isolate from France

ABSTRACT An NDM-1 carbapenemase-producing Pseudomonas aeruginosa isolate was recovered from a patient hospitalized in France after a previous hospitalization in Serbia. Genetic studies revealed that the blaNDM-1 gene was surrounded by insertion sequence ISAba125 and a truncated bleomycin resistance gene. This blaNDM-1 region was a part of the variable region of a new complex class 1 integron bearing IS common region 1 (ISCR1). The presence of ISPa7 upstream of this integron suggests insertion in a chromosomally located Tn402-like structure.

Characterization of a Novel BCHE “Silent” Allele: Point Mutation (p.Val204Asp) Causes Loss of Activity and Prolonged Apnea with Suxamethonium

Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of suxamethonium leading to the discovery of a novel BCHE variant (c.695T>A, p.Val204Asp). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation disrupts the catalytic triad and determines a “silent” phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC) and benzoylcholine, and values of dibucaine and fluoride numbers fit with heterozygous atypical silent genotype. Electrophoretic analysis of plasma BChE of the proband and his mother showed that patient has a reduced amount of tetrameric enzyme in plasma and that minor fast-moving BChE components: monomer, dimer, and monomer-albumin conjugate are missing. Kinetic analysis showed that the p.Val204Asp/p.Asp70Gly-p.Ala539Thr BChE displays a pure Michaelian behavior with BTC as the substrate. Both catalytic parameters Km = 265 µM for BTC, two times higher than that of the atypical enzyme, and a low Vmax are consistent with the absence of activity against suxamethonium. Molecular dynamic (MD) simulations showed that the overall effect of the mutation p.Val204Asp is disruption of hydrogen bonding between Gln223 and Glu441, leading Ser198 and His438 to move away from each other with subsequent disruption of the catalytic triad functionality regardless of the type of substrate. MD also showed that the enzyme volume is increased, suggesting a pre-denaturation state. This fits with the reduced concentration of p.Ala204Asp/p.Asp70Gly-p.Ala539Thr tetrameric enzyme in the plasma and non-detectable fast moving-bands on electrophoresis gels.

Analytical performances of the Diazyme ADA assay on the Cobas® 6000 system

OBJECTIVE To evaluate the analytical performance of the Diazyme ADA assay on the Cobas® 6000 system for pleural fluid samples analysis. DESIGN AND METHODS Imprecision, linearity, calibration curve stability, interference, and correlation studies were completed. RESULTS The Diazyme ADA assay demonstrated excellent precision (CV<4%) over the analytical measurement range (0.5-117 U/L). Bilirubin above 50 μmol/L and haemoglobin above 177 μmol/L interfered with the test, inducing a negative and a positive interference respectively. The Diazyme ADA assay correlated well with the Giusti method (r(2)=0.93) but exhibited a negative bias (~ -30%). CONCLUSIONS The Diazyme ADA assay on the Cobas® 6000 system represents a rapid, accurate, precise and reliable method for determination of ADA activity in pleural fluid samples.

Pseudomonas aeruginosa et résistance aux antibiotiques

Resume L’acquisition de resistances aux β-lactamines chez Pseudomonas aeruginosa resulte de mutations entrainant une surproduction de la cephalosporinase constitutive AmpC, une surexpression des systemes d’efflux actif, une diminution de permeabilite membranaire et l’acquisition de genes exogenes. La dissemination de β-lactamases a spectre etendu, de metallo-carbapenemases et d’oxacillinases a spectre elargi est un phenomene emergent que le biologiste doit pouvoir rapidement evoquer. La resistance aux aminosides est egalement frequente, liee a l’acquisition d’enzymes modificatrices ou de methylases et parfois d’une surexpression de pompes d’efflux. La resistance aux fluoroquinolones est essentiellement liee a des mutations dans la sous-unite GyrA de l’ADN gyrase. La surexpression des pompes d’efflux contribue egalement a cette resistance. L’accumulation de ces differents mecanismes peut conduire a des souches de Pseudomonas aeruginosa multiresistantes ou toto-resistantes, comme ceci est decrit de plus en plus frequemment dans le monde, notamment dans les unites de soins intensifs. Les auteurs exposent les grandes tendances de l’epidemiologie de la resistance aux antibiotiques pour cette espece, les recommandations pour la determination de la sensibilite in vitro au laboratoire, ainsi que les differents outils disponibles pour la detection des β-lactamases a spectre etendu, de metallo-carbapenemases et d’oxacillinases a spectre elargi.

Abdominal wall and intra-peritoneal abscess by Propionibacterium avidum as a complication of abdominal parietoplasty.

Propionibacteria are organisms of low pathogenicity and only a minority of clinical Propionibacterium isolates is clinically significant. Herein, we report a rare case of Propionibacterium avidum abdominal wall and intra-peritoneal abscess that developed in 46-year-old woman after abdominal parietoplasty.

Efficacy of a swab transport system in maintaining long-term viability of Staphylococcus aureus

The noncharcoal liquid Amies swab transport system (Copan, Bovezzo, Italy) can be used for the collection and transport of biologic samples from carriers and infected patients for the detection of strains of Staphylococcus aureus in epidemiologic field studies. We suggest that the maximum holding time, between swab collection and culture, should be 18 days.

The C5 Variant of the Butyrylcholinesterase Tetramer Includes a Noncovalently Bound 60 kDa Lamellipodin Fragment

Humans with the C5 genetic variant of butyrylcholinesterase (BChE) have 30–200% higher plasma BChE activity, low body weight, and shorter duration of action of the muscle relaxant succinylcholine. The C5 variant has an extra, slow-moving band of BChE activity on native polyacrylamide gel electrophoresis. This band is about 60 kDa larger than wild-type BChE. Umbilical cord BChE in 100% of newborn babies has a C5-like band. Our goal was to identify the unknown, 60 kDa protein in C5. Both wild-type and C5 BChE are under the genetic control of two independent loci, the BCHE gene on Chr 3q26.1 and the RAPH1 (lamellipodin) gene on Chr 2q33. Wild-type BChE tetramers are assembled around a 3 kDa polyproline peptide from lamellipodin. Western blot of boiled C5 and cord BChE showed a positive response with an antibody to the C-terminus of lamellipodin. The C-terminal exon of lamellipodin is about 60 kDa including an N-terminal polyproline. We propose that the unknown protein in C5 and cord BChE is encoded by the last exon of the RAPH1 gene. In 90% of the population, the 60 kDa fragment is shortened to 3 kDa during maturation to adulthood, leaving only 10% of adults with C5 BChE.

Adenosine deaminase is a useful biomarker to diagnose pleural tuberculosis in low to medium prevalence settings

Adenosine deaminase (ADA) activity measurement in pleural fluid is a relevant test to diagnose pleural tuberculosis (pTB) in high tuberculosis prevalence settings. We investigated the diagnostic utility of pleural ADA using a retrospective analysis of patients admitted with newly diagnosed pleural effusion without identified etiology between 2001 and 2008 in Paris suburb, a low to medium tuberculosis prevalence area. 104 adults (mean age 55 years; 34 with pTB, 70 with other diagnoses) were analyzed. Median follow-up was 15.6 months. Mean [interquartile range] pleural ADA was 119 U/L [IQR: 83-143] in pTB and 24 U/L [IQR: 15-31] in non-tuberculous effusions (P<0.001). With an optimal pleural ADA cut-off value of 41.5 U/L for pTB diagnosis, sensitivity and specificity were 97.1% and 92.9%, while positive and negative predictive values were 86.8% and 98.5%, respectively. We conclude that pleural ADA activity could be integrated in the diagnostic procedures of pTB in low to medium tuberculosis prevalence settings.

Déficit génétique en butyrylcholinestérase

Butyrylcholinesterase (EC 3.1.1.8; BChE) is a sister enzyme of acetylcholinesterase. Though BChE lacks obvious physiological functions, it is of toxicological and pharmacological importance in detoxifying or catabolising ester-containing drugs. Furthermore, individuals deficient in BChE appear asymptomatic, apart from a heightened sensitivity to the muscle relaxants suxamethonium and mivacurium, two BChE substrates used as myorelaxant. Although many acquired conditions may affect BChE activity, BChE deficiency is mainly due to mutations in the BCHE gene (OMIM 177400). Currently, more than 70 natural mutations have been documented in human BCHE. They have an adverse effect on BChE activity by affecting the catalytic functioning or the protein expression. However, the atypical variant (rs1799807) is the most frequently involved in prolonged apnea.

Pleural adenosine deaminase determination: an inter-laboratory comparison is required.

We read with interest the article of Keng et al. about the interest of various biomarkers for diagnosing tuberculous pleurisy (TB). In accordance with previous studies, the authors concluded that pleural adenosine deaminase (ADA) determination is a useful tool. However, they reported an optimal cut-off value significantly lower than those commonly mentioned in the literature (15.5 IU/L versus 30 or 40 IU/L) and especially in another study performed in the same country (cut-off: 30 IU/L). The authors hypothesized that this difference may be related to either the study population (i.e., TB patients older than those of the other Taiwanese study), or to the methodology used for pleural ADA determination. As underlined in the article, the laboratory methodologies, such as Giusti or non-Giusti methods, have a significant impact on the pleural ADA determination. Recently, we have reported a lack of transferability between pleural ADA assays. The two studies performed in Taiwan used the same commercial kit (Adenosine Deaminase Assay Kit, Diazyme Laboratories, San Diego, CA, USA). This kit can be adapted to various analytical systems. For example, in our daily practice, we have adapted it on the Cobas 6000 system (Roche Diagnostics, GmbH, Mannheim, Germany) with a cut-off value of 30 IU/L. It cannot be excluded that the analytical system on which the kit is adapted can impact the inter-studies comparisons. To confirm or infirm this hypothesis, it would be interesting to perform an interlaboratory comparison to determine the potential impact of the analytical systems on ADA determination with the Diazyme Laboratories’ kit. Based on the results obtained, specific cut-off values could be proposed depending on the analytical system or on the age of the patients.