Naked-eye nanobiosensor for therapeutic drug monitoring of methotrexate.

Sensing of methotrexate at clinically-relevant concentrations was achieved with a plasmon-coupling assay. In this assay, free methotrexate and folic acid Au nanoparticles competed for human dihydrofolate reductase (hDHFR)-functionalized Au nanoparticles (Au NP). The hDHFR-functionalized Au NPs were immobilized on a small glass sensor inserted in a portable 4-channel LSPR reader. This allowed rapid (minutes) and sensitive (nanomolar range) measurement of methotrexate concentration by means of total internal reflection plasmonic spectroscopy. The large bathochromic shifts of the plasmon-coupling assay led to striking colour changes visible to the naked eye for methotrexate at clinically-relevant concentrations. The results demonstrate the potential for therapeutic drug monitoring of a widely used chemotherapy agent, as assessed with the naked eye.

Plasmonic sensors for the competitive detection of testosterone.

The ability to detect small molecules in a rapid and sensitive manner is of great importance in the field of clinical chemistry, and the advancement of novel biosensors is key to realising point-of-care analysis for essential targets. Testosterone is an example of such a small molecule, the detection of which is important in both clinical analysis, and in the sporting industry to prevent doping. As such, a portable, rapid and sensitive test for testosterone would be of great use across a variety of analytical fields. Here we report on a novel method of testosterone analysis, based on a competitive inhibition assay utilising functionalized gold nanoparticles. Two sensing platforms are directly compared for the detection of testosterone based on both classical SPR and LSPR. We provide an in-depth discussion on the optimum surface chemistries needed to create a stable detection conjugate before successfully detecting testosterone using our newly developed portable 4-channel SPR instrument. We provide the first detailed study into the comparison of SPR and LSPR for the analysis of a small molecule, and provide a simple and effective method of testosterone detection that could potentially be extended to a variety of different analytes.

Thrombocytopenia induced by the histone deacetylase inhibitor abexinostat involves p53-dependent and -independent mechanisms.

Abexinostat is a pan histone deacetylase inhibitor (HDACi) that demonstrates efficacy in malignancy treatment. Like other HDACi, this drug induces a profound thrombocytopenia whose mechanism is only partially understood. We have analyzed its effect at doses reached in patient plasma on in vitro megakaryopoiesis derived from human CD34(+) cells. When added at day 0 in culture, abexinostat inhibited CFU-MK growth, megakaryocyte (MK) proliferation and differentiation. These effects required only a short incubation period. Decreased proliferation was due to induction of apoptosis and was not related to a defect in TPO/MPL/JAK2/STAT signaling. When added later (day 8), the compound induced a dose-dependent decrease (up to 10-fold) in proplatelet (PPT) formation. Gene profiling from MK revealed a silencing in the expression of DNA repair genes with a marked RAD51 decrease at protein level. DNA double-strand breaks were increased as attested by elevated γH2AX phosphorylation level. Moreover, ATM was phosphorylated leading to p53 stabilization and increased BAX and p21 expression. The use of a p53 shRNA rescued apoptosis, and only partially the defect in PPT formation. These results suggest that HDACi induces a thrombocytopenia by a p53-dependent mechanism along MK differentiation and a p53-dependent and -independent mechanism for PPT formation.

Myeloproliferative disorders: the centrosome connection.

Some myeloproliferative disorders (MPD) result from a reciprocal translocation that involves the FGFR1 gene and a partner gene. The event creates a chimeric gene that encodes a fusion protein with constitutive FGFR1 tyrosine kinase activity. FGFR1-MPD is a rare disease, but its study may provide interesting clues on different processes such as cell signalling, oncogenesis and stem cell renewal. Some partners of FGFR1 are centrosomal proteins. The corresponding oncogenic fusion kinases are targeted to the centrosome. Constitutive phosphorylation at this site may perturbate centrosome function and the cell cycle. Direct attack at this small organelle may be an efficient way for oncogenes to alter regulation of signalling for proliferation and survival and get rid of checkpoints in cell cycle progression. The same effect might be triggered by other fusion kinases in other MPD and non-MPD malignancies.

[Sequelae of intra-articular calcaneal fractures: patterns and management]. / Chirurgie des séquelles des fractures du calcanéum: à propos de 24 cas.

PURPOSE OF THE STUDY: We reviewed retrospectively 24 feet with sequelae of transtalar process fractures of the calcaneum in order to identify the lesion pattern and determine optimal management options, both for acute and sequelar lesions. MATERIAL AND METHODS: There were fourteen men and nine women, mean age 42 years (19-73). Twenty-three had subtalar osteoarthritis, eight had calcaneocuboid osteoarthritis, and fifteen had lateral submalleolar conflicts. There were twelve fibular tendon dislocations or fissurations, three tarsal tunnel syndromes, and two plantar splinters. Prior to treatment, all patients complained of pain. Preoperatively, walking distance was less than 500 m for thirteen patients, 2000-3000 m for four, and greater than 3000 m for five. Mean subtalar joint motion was 30% (0-100%) compared with the healthy side and mean frontal misalignment of the rear foot was 6 degrees valgus. Physical examination, podoscopy and x-rays were obtained in all patients. The Kitaoka score was noted. RESULT: Mean follow-up was 36 months (24-72). Sequelae were treated with a single procedure or with combinations: subtalar arthrodesis (n = 23) including one in association with calcaneocuboid arthrodesis, tension on fibular tendons (n = 7), neurolysis of the posterior tibial nerve (n = 3), resection of plantar splinters (n = 2), resection of the lateral shell (n = 14), and osteotomy (n = 2) to lower the greater tubercle of the calcaneum because of pain when wearing shoes. The mean Kitaoka function score was 31.7/100 (14-79) preoperatively. After treatment, the mean score was 81.7/100 (31-94), giving a 73.2% gain. The outcome was considered good in sixteen feet, fair in six, and poor in two. Mean walking distance was greater than 3000 m for 18 patients. Mean frontal misalignment of the rear foot under loading was 4.5 degrees valgus and the podoscopy demonstrated flat foot in thirteen patients. Three subtalar arthrodesis required revision for nonhealing. DISCUSSION: Initial treatment of a fracture, particularly an articular fracture, of the calcaneum must avoid disabling postoperative pain and shoe wearing problems. These sequelae basically concern: subtalar and calcaneocuboid arthritis, lateral submalleolar conflict, fibular tendon injury, plantar splinters, tarsal tunnel syndrome, loss of height, and misalignment of the rear foot. At the sequelar stage, the physical examination is primordial to confirm the lesion and search for any complication which could develop later postoperatively when walking distance becomes longer. For nine patients with residual pain, four resulted from lesions which were missed at the preoperative physical examination. Arthrodesis of the subtalar joint should be preferred over realignment of the rear foot and can be associated with the treatment of conflicts. This management scheme allows treating during a single operative time all sequelae, thus limiting recovery time. A scan of the ankle and foot with or without opacification of the fibular tendons is needed to confirm the physical examination which, for us, remains the key to successful surgery.

[Rhône-Alpes observatory of Streptococcus pneumoniae in 1999: 35 cases of meningitis]. / Observatoire Rhône-Alpes du pneumocoque en 1999: 35 cas de méningites.

In 1999, in Rhône-Alpes region, in a survey of resistance to antibiotics of Streptococcus pneumoniae, 35 cases of meningitis were observed. A retrospectic questionnary was sent to each participant. MICs to Penicillin, Amoxicillin and Cefotaxime were determined with ATB-PNEUMO gallery or E-test and by disk diffusion for the other antibiotics. The results were interpreted according to the recommendations of the CA-SFM. Mean age was 38.1 years (range : 1 month -78 years) and sex-ratio 2/5. Eight patients had previously received antibiotics, 22 patients had risk factors and 23 were transferred in intensive care unit. The patients received C3G + glycopeptide in 15 of 16 children and in 13/19 adults according to the consensus recommendations. Diagnostic was made on the direct examination of CSF in 83%, and blood cultures was positive in 74.3% of cases. The percentage of PRP was 48.6% with 17.1% of intermediate-amoxicilline and 14.3% intermediate-cefotaxime strains. Resistance to trimethoprim-sulfamethoxazole was 45.7%, to chloramphenicol 30% and to fosfomycin 6.9%. All the strains were susceptible to rifampicin and vancomycin. Among the 17 PRP strains, 7 were belonging to serotype 6 (6 in children). The clinical outcome was fatal in 7 male cases (20%), without risk factors in 3 children and 6 of 7 strains were susceptible to penicillin. Six patients (17%) had auditive and/or neurologic sequellaes. This study shows that nearly 50% of strains isolated in meningitis, in Rhône-Alpes region, were not susceptible to penicillin, and confirms the frequency of sequellaes while the mortality is not related with the resistance of strains to the antibiotics.

Comparative in vitro activity of penicillin G, levofloxacin, moxifloxacin, telithromycin, pristinamycin, quinupristin-dalfopristin and linezolid against ofloxacin-intermediate and -resistant Streptococcus pneumoniae.

Screening by ofloxacin disk was carried out on 1158 strains of Streptococcus pneumoniae in order to investigate the in vitro bacteriostatic activity of penicillin G, levofloxacin, moxifloxacin, telithromycin, linezolid, pristinamycin and quinupristin-dalfopristin against ofloxacin-intermediate and -resistant S. pneumoniae strains. It was concluded that these new antimicrobial agents could be useful for the treatment of pneumococcal infections caused by penicillin-sensitive and -resistant S. pneumoniae, and would represent a valid therapeutic option for patients allergic to beta-lactams, should they prove to be potent in vivo.

[Evaluation of the E-test and the ATB-PNEUMo battery for determining the beta-lactam MIC for Streptococcus pneumoniae in daily practice]. / Evaluation du E-test et de la galerie ATB-PNEUMo dans la détermination des CMI aux bêtalactamines de Streptococcus pneumoniae en pratique quotidienne.

In 1999, during the survey of resistance of Streptococcus pneumoniae to antibiotics by 31 clinical laboratories of Rhône-Alpes area, MIC to penicillin (P), amoxicillin (AMX) and cefotaxime (CTX) of 877 PRP strains or with a diameter of inhibition to oxacillin inferior to 26 mm, were determined by each institution by E-test (n = 220 strains) or ATB-PNEUMO (n = 657 strains). MICs of these three antibiotics were determined by dilution in agar medium by the coordinating center. The essential agreement was respectively for ATB-PNEUMO and E-test 89% versus 84% for P (p > 0.05), of 86% vs 79% for AMX (p < 0.01), and of 91% vs 86% for CTX (p = 0.03). When the strains were classified in clinical category, the differences were significant (p < 0.001) for AMX (85% vs 71%) and for CTX (82% vs 75%) but not for P (73% vs 78%). ATB-PNEUMO method was more sensitive than E-test for the detection of strains susceptible to P (90 vs 73%), to AMX (83 vs 78%) and to CTX (80 vs 72%) and for the strains intermediate to AMX (90 vs 78%). On the contrary, E-test is more specific than ATB-PNEUMO for the detection of P-resistant strains (94 vs 86%). Finally, the specificity of both methods is the same for detection of P-S, AMX-R and CTX-I strains.

Fitness and competitive growth advantage of new gentamicin-susceptible MRSA clones spreading in French hospitals.

Since 1991, new epidemic methicillin-resistant Staphylococcus aureus (MRSA) strains characterized by the unexpected reappearance of heterogeneous phenotypic expression of resistance to methicillin and by susceptibility to gentamicin and various other antibiotics (GS-MRSA) have been reported in France. GS-MRSA strains have progressively replaced MRSA clones expressing homogeneous resistance to methicillin and resistance to gentamicin (GR-MRSA). In this study, we investigated the physiological characteristics of these new clones. In particular, we evaluated and compared the maximal growth rate and the deduced generation times (related to fitness of strains) of the major French epidemic MRSA clones. The population studied consisted of 79 isolates including (i) GR-MRSA that comprised six different types on the basis of PFGE; (ii) GS-MRSA the majority of which clustered into two PFGE types, A1 (usually resistant to erythromycin) and B (usually susceptible to erythromycin); (iii) methicillin-susceptible S. aureus (MSSA). GS-MRSA-A1 and MSSA strains were shown to have a significant fitness benefit (about 20%) with shorter generation times (theta = 23.7 +/- 0.1 and 22.9 +/- 0.05 min, respectively) than GR-MRSA and GS-MRSA-B strains (theta = 30.3 +/- 0.2 and 32.5 +/- 0.5 min, respectively). These data suggest that a link exists between genetic patterns, resistance profiles and physiological properties. In vitro competitive experiments indicated that GS-MRSA- A1 strains were able to rapidly outgrow GR-MRSA strains. The growth advantage observed should be taken into account in understanding the spread of some new clones of MRSA.

[Pasteurella multocida lung abscess. Study of a case with demonstration of a cat as vector]. / Pneumopathie abcédée à Pasteurella multocida. Etude d'un cas avec démonstration du rôle du chat comme vecteur.

We report a case of a lung abscess due to Pasteurella multocida, isolated from bronchial secretions by fiberoptic bronchoscopy. Antibodies against Pasteurella multocida were elevated in the patient, and absent in controls (including the patient's wife). The identity of the strain isolated in the patient and that in his cat was proved by molecular method using pulsed field gel electrophoresis.

Zero rate of shunt infection in the first postoperative year in children--dream or reality?

The rate of infectious complications following shunt implantations at the Pierre Wertheimer Hospital was 6.4% in 1992-1994. In order to improve this infection rate, new recommendations for surgery were applied and a new type of valve was used. The effects of these measures after a 1-year follow-up were analyzed in 70 patients. The rate of infection was zero, 2.8, and 4.3% at 2, 6, and 12 months, respectively. A case-control study did not reveal any significant risk factor among the patient and surgical factors analyzed.

Emergence and spread in French hospitals of methicillin-resistant Staphylococcus aureus with increasing susceptibility to gentamicin and other antibiotics.

Oxacillin (methicillin) resistance in methicillin-resistant Staphylococcus aureus (MRSA) is associated with an increased incidence of resistance to other antibiotics, which has increased since it was first reported in 1969. In 1992 a new phenotype of MRSA arose in France; this was characterized by a heterogeneous expression of resistance to oxacillin and susceptibility to various antibiotics, including gentamicin but also tetracycline, minocycline, lincomycin, pristinamycin, co-trimoxazole, rifampin, and fusidic acid. In French hospitals a longitudinal nationwide surveillance of antibiotic resistance in S. aureus has allowed for the detection of changes in antibiotic susceptibility profiles. Seven French clinical laboratories (six from the mainland and one from the West Indies) reported the results of susceptibility testing of 57,347 S. aureus strains isolated in their institutes between 1992 and 1998. Over a 7-year period the incidence of isolation of gentamicin-susceptible MRSA (GS-MRSA) strains has steadily increased to represent, in 1998, 46.8 to 94.4% of the MRSA strains, irrespective of the overall incidence of MRSA. Two predominant types recognized by pulsed-field gel electrophoresis (PFGE) accounted for the majority of the GS-MRSA in different mainland hospitals, both differing from the predominant type observed in the French West Indies. Some GS-MRSA and gentamicin-resistant MRSA (GR-MRSA) strains had closely related PFGE profiles, and hybridization studies confirmed the lack in GS-MRSA of the aac6'-aph2" gene, which confers resistance to all aminoglycosides, with conservation of the ant4' gene, which confers resistance to kanamycin, tobramycin, and amikacin. Thus, it is likely that certain GS-MRSA strains could have emerged from GR-MRSA strains by excision or deletion of the aac6'-aph2" gene.

[Pneumococcus observatory data in the Rhône-Alps region. Results from 1996]. / Observatoire du pneumocoque en région Rhône-Alpes. Résultats de l'année 1996.

Throughout 1996, 22 hospital-based laboratories in the Rhône-Alpes region of France collected pneumococcal strains and used a standardized protocol to record the following data; patient age and sex; type of specimen; and determination of susceptibility to at least the following antibiotics: oxacillin 1 microgram and 5 micrograms, erythromycin (Ery), tetracycline (Tet), chloramphenicol (Chl), rifampin (Rmp), and loracarbef. For penicillin-nonsusceptible strains (PNSSs), which were identified based on results with oxacillin, MICs for penicillin G, amoxicillin (Amx), and cefotaxime (Ctx) were determined using the E Test, at the study site and agar dilution at the coordinating center. Of the 1153 strains, 65.5% were from adults and 31.8% from children; patient age was unknown in 2.7% of cases. PNSPs (MIC > 0.06 mg/l) contributed 32.9% of strains (I: 23.3%; R: 9.6%) and were more common in children (41.1%) than in adults (28.1%). The frequency of PNSSs varied across specimen types: 27.9% in blood cultures (305 strains), 15.6% in cerebrospinal fluid (32), 38.7% in protected bronchopulmonary specimens (31), 31.5% in unprotected bronchopulmonary specimens (434), 50.8% in acute otitis media (118), and 34.4% in other specimens (221). Among PNSSs, nonsusceptibility (I + R) to other antibiotics was variable: Ery, 62.1%; Tet, 41.5%; Chl, 40.4%; Rmp, 1.1%. Corresponding figures for the overall strain population were Ery, 33.3%; Tet, 22.7%; Chl, 22.8%; Rmp, 0.9%. In addition, 56.5% of PNSSs exhibited multiple drug resistance. Resistance to amoxicillin (MIC > 2 mg/l) was demonstrated for only 5 strains. No strains were resistant to loracarbef or cefotaxime. Serotypes of the 379 PNSSs were as follows: 23F, 26.6%; 14 (25.6%); 9V (18.2%), 6 (8.7%), 15 (5%), 19 (4.5%).

Role of the preaxillary flora in pacemaker infections: a prospective study.

BACKGROUND: Infection remains a severe complication after pacemaker implantation. The purpose of our prospective study was to evaluate the role of the local bacteriologic flora in its occurrence. METHODS AND RESULTS: Specimens were collected at the site of implantation for culture from the skin and the pocket before and after insertion in a consecutive series of patients who underwent elective permanent pacemaker implantation. Microorganisms isolated both at the time of insertion and of any potentially infective complication were compared by using conventional speciation and ribotyping. There were 103 patients (67 men and 36 women) whose age ranged from 16 to 93 years (mean+/-SD, 67+/-15). At the time of pacemaker implantation, a total of 267 isolates were identified. The majority (85%) were staphylococci. During a mean follow-up of 16.5 months (range, 1 to 24), infection occurred in four patients (3.9%). In two of them, an isolate of Staphylococcus schleiferi was recognized by molecular method as identical to the one previously found in the pacemaker pocket. In one patient, Staphylococcus aureus, an organism that was absent at the time of pacemaker insertion, was isolated. In another patient, a Staphylococcus epidermidis was identified both at the time of pacemaker insertion and when erosion occurred; however, their antibiotic resistance profiles were different. CONCLUSIONS: This study strongly supports the hypothesis that pacemaker-related infections are mainly due to local contamination during implantation. S schleiferi appears to play an underestimated role in infectious colonization of implanted biomaterials and should be regarded as an important opportunistic pathogen.

Multicenter clinical comparison of resin-containing bottles with standard aerobic and anaerobic bottles for culture of microorganisms from blood.

In a study comparing the Bactec 9240 (Plus Aerobic/F and Anaerobic/F bottles, containing resins; Becton Dickinson, USA) and the Vital (standard aerobic and anaerobic bottles, with no additives; bioMérieux, France) blood culture systems, 6456 sets of four bottles of 9660 blood cultures submitted were evaluated. There were 531 clinically significant isolates from 795 positive blood cultures. Of the 531 positive blood cultures, 355 were positive in both systems, 141 with the Bactec 9240 alone, and 30 with the Vital alone (p < 0.001); five were not detected by either system. The average time to detection of positive cultures for the matched sets was 10.65 h and 18.41 h by the Bactec 9240 system and the Vital system, respectively. The false-positive rate per bottle was 0.65% in the Bactec 9240 and 0.71% in the Vital. The rate of false-negative pairs (i.e., major errors) was very low (0.12% for the Bactec 9240, 0.19% for the Vital) and not significantly different between the two systems. The striking differences in recovery of microorganisms may be due to the presence of resins in the Bactec medium. However, the observed superiority of the Bactec 9240, even for patients not receiving antibiotics, suggests that resins adsorb other inhibitors present in patients' blood.

Evaluation of a commercial test kit for rapid detection of Staphylococcus aureus in blood cultures.

The presence of Staphylococcus aureus in positive blood cultures could be rapidly confirmed by the use of a commercial test kit (Rapidec Staph; bioMérieux, France) that detects coagulase in the culture broth after 2 h of incubation. This kit detected all 35 Staphylococcus aureus strains from 117 blood cultures with gram-positive cocci growing in clusters. The sensitivity and the specificity of the technique were 100% and 98.8%, respectively. The Rapidec Staph kit appears to be a simple and reliable technique for the rapid detection of Staphylococcus aureus in blood cultures.

AUDILAB: quality audit simulation to prepare biomedical testing laboratories for accreditation.

In order to prepare biomedical laboratories for accreditation a quality audit simulator has been developed. By means of a dialogue with the laboratory, AUDILAB simulates the process by which the auditor identifies deviations or assesses conformity to the standard. AUDILAB is based on a knowledge processing system accessible via Minitel (French videotex terminal). With reference to the EN 45001 standard which is the European standard concerning the "General criteria for the operation of testing laboratories", it provides an assessment of the laboratory's degree of conformity and prompts it to take corrective action.