Increased Performances of the Biological Diagnosis of the Antiphospholipid Syndrome by the Use of a Multiplex Assay.

Antiphospholipid syndrome (APS) is characterized by development of venous and/or arterial thrombosis and pregnancy morbidity. Biological criteria are the persistent presence of lupus anticoagulant (LA) and/or anti-cardiolipin (aCL) and/or anti-B2GP1 autoantibodies' positivity. The assays' performances are of crucial importance. We evaluated a multiplex assay allowing simultaneous detection of IgG anti-cardiolipin, anti-B2GP1, and anti-factor II. 300 samples were tested. Patients were categorized according to clinical scores of APS from 0 to 3 based on presence or not of arterial or venous thrombosis, fetal loss, and autoimmunity. We used a multiplex assay for APS for simultaneous detection of aCL, anti-B2GP1, and factor II and compared its performances to ELISA assays. Presence of LA was also assessed. We performed a correlation study of the tested assays and compared their clinical efficacy by ROC curve analysis. We obtained significantly higher performances with the multiplex assay than ELISA with higher area under the curve (AUC). The disease rate increased with the number of positive markers from 9% for 1 marker to 100% for 4 markers positive for patients with high risk scores. The multiplex APS assay exhibited higher performances particularly in case of primary APS and is useful for rapid diagnosis of APS.

Fighting the spread of AmpC-hyperproducing Enterobacteriaceae: beneficial effect of replacing ceftriaxone with cefotaxime.

OBJECTIVES: Considering the hypothesis that the high biliary elimination of ceftriaxone could be responsible for the selection of Enterobacteriaceae harbouring high-level AmpC ß-lactamase (HL-CASE), the use of ceftriaxone was discontinued in our hospital in 2006 and replaced with cefotaxime. METHODS: Antibiotic consumption, expressed as defined daily dose (DDD)/1000 patient-days (PD), and HL-CASE incidence, expressed as the number of patients carrying HL-CASE/1000 PD, were compared between the pre-intervention period (Period 1, 2001-05) and the post-intervention period (Period 2, 2006-12) using an interrupted time series analysis. RESULTS: The incidence of HL-CASE increased significantly from 0.32 to 0.69/1000 PD during Period 1 (coefficient = 0.082, P < 0.01). A significant inflection of the slope in the incidence curve occurred in Period 2 (coefficient = -0.061, P = 0.05), mainly owing to the stabilization of the HL-CASE incidence of Enterobacteriaceae harbouring chromosomally inducible cephalosporinase (Period 1, 0.27 to 0.64/1000 PD; Period 2, 0.58 to 0.61/1000 PD) and especially for Enterobacter cloacae (Period 1, 0.09 to 0.30/1000 PD; Period 2, 0.26 to 0.27/1000 PD). This deceleration was observed despite a significant increase in the slope of cefotaxime consumption over Period 2 (coefficient = 2.97, P < 0.01). CONCLUSION: Despite the disadvantages of using cefotaxime compared with ceftriaxone (administration three times daily versus once a day), the ecological benefits of this substitution seem sufficiently convincing to preferentially use cefotaxime. Control of HL-CASE incidence is crucial to limiting carbapenem use and preventing the selection of carbapenemase-producing Enterobacteriaceae.

Comparison of CD63 Upregulation Induced by NSAIDs on Basophils and Monocytes in Patients with NSAID Hypersensitivity.

Background. An in vitro basophil activation test, based on the detection of CD63 upregulation induced by NSAIDs, has been described. Its clinical significance remains controversial. Objectives. In patients with a history of nonallergic NSAID hypersensitivity, stratified according to the severity of the symptoms, to assess with NSAIDs the predictive value of basophil (BAT) and monocyte (MAT) activation tests. Patients/Methods. Sixty patients who had NSAIDs-induced or exacerbated urticaria/angiooedema and 20 controls was included. After incubation with NSAIDs or acetaminophen, leukocytes were analysed for CD63 upregulation. Results. With aspirin, the sensitivity (37%) and specificity (90%) of BAT agree with already published results. In contrast, when patients had had cutaneous and visceral reactions, the frequency of positive BAT 14/22 (64%, P < 0.001) or MAT 10/22 (46%, P < 0.01) were increased. Conclusions. Positive tests were more frequent among patients having a severe hypersensitivity contrasting with the other patients who had results similar to controls.

Comparison of two basophil activation markers CD63 and CD203c in the diagnosis of amoxicillin allergy.

BACKGROUND: To confirm allergy to beta-lactam (BL), a basophil activation test in flow cytometry based on CD63 up-regulation was described. CD203c is a more recent basophil activation marker and up to day there is no consensus about which marker is the more sensitive one. CD203c has not yet been evaluated in the diagnosis of BL allergy. OBJECTIVE: The aim of the study was to compare the reliability of CD203c to CD63 for the diagnosis of amoxicillin (AX) allergy, which is nowadays the most frequent BL allergy. METHODS: Twenty-seven patients with an immediate positive skin test (ST) to AX, 20 had had anaphylaxis with AX and 7 had urticaria and/or angioedema, were compared with 14 controls with no allergy to BL and to six patients with delayed positive ST to AX. RESULTS: In the anaphylaxis group, AX induced up-regulation of CD203c in the basophils of 12 patients out of 20 (60%) and of CD63 in four patients (20%) (P<0.02). Two patients out of seven with urticaria or angioedema had a positive result with CD203c and CD63. In patients who had anaphylaxis, ampicillin (AMP) induced CD203c up-regulation in eight out of 12 (67%) patients tested, and CD63 up-regulation in 4 out of 12 (33%) (all patients who had anaphylaxis could not be tested with AMP). False-positive results were observed with CD203c as well as CD63, and for 10 patients indeed this was confirmed by a negative drug provocation test. The origin of conflicting results between CD63 and CD203c might be at least the targeting of basophils based on anti-IgE labelling. Among IgE(+) gated cells, by means of CD33, a marker of monocytes, a contamination up to 50% by monocytes was detected. In contrast to CD63, CD203c is an activation marker specific of basophils with a basal low-level expression in resting basophils. Thus, IgE and CD203c double targeting of basophils avoids the contamination by monocytes. CONCLUSION: CD203c seems to be a more sensitive activation marker of basophils than CD63 for the diagnosis of amoxicillin allergy.

Effects of topical anaesthetics and fluorescein on the real-time PCR used for the diagnosis of Herpesviruses and Acanthamoeba keratitis.

BACKGROUND: The early microbiological diagnosis of corneal infections may prevent the condition from worsening. AIM: To study the potential interferences of oxybuprocain and fluorescein solutions used by ophthalmologists on the performances of the real-time polymerase chain reaction (PCR) carried out as routine test for diagnosis of keratitis. METHODS: Quantified suspensions of Herpes simplex virus (HSV1), Varicella zoster virus (VZV), Cytomegalovirus (CMV) and Acanthamoeba with and without oxybuprocain or fluorescein added before DNA extraction were tested by real-time PCR. RESULTS: The capacities of the real-time PCR to detect HSV, VZV, CMV and Acanthamoeba were reduced by oxybuprocain and fluorescein. Both products diluted to 1/16 reduced the PCR detection capacities for more than 2 logs (DNA copies/sample). CONCLUSIONS: The simultaneous introduction of fluorescein or topical anaesthetics into the tubes containing the specimens to be tested by PCR may lead to false negative results. Because corneal specimens for microbiological diagnosis of keratitis are obtained after topical administration of anaesthetics and corneal staining with fluorescein, ophthalmologists should be aware to rinse the eye surface intensively with appropriate eye solutions to minimise the risks of misdiagnosis.

Detection by broad-range real-time PCR assay of Chlamydia species infecting human and animals.

BACKGROUND: Tests available for molecular diagnosis of chlamydial infections detect Chlamydiatrachomatis, but do not find other Chlamydia species associated with genital, ophthalmic, cardiovascular, respiratory or neurological diseases. The routine detection of all Chlamydia species would improve the prognosis of infected people and guide therapeutic choices. AIM: To design and validate a sensitive, specific, reproducible, inexpensive and easy-to-perform assay to quantify most Chlamydia species. METHODS: Primers and probe were selected using the gene coding for the 16S rRNA. The detection limits were assessed for suspensions of Chlamydia trachomatis, Chlamydia psittaci and Chlamydia pneumoniae. The performance of this test was compared with that of two commercial kits (Amplicor-Roche and Artus) on 100 samples obtained from children with trachoma. RESULTS: The detection capacities for Chlamydia trachomatis of the broad-range real-time polymerase chain reaction (PCR) were similar or slightly better than those obtained with commercial kits (0.2 copies of DNA/microl). Only the broad-range PCR identified specimens containing Chlamydia psittaci and Chlamydia pneumoniae. The commercial kits and the broad-range assay detected Chlamydia species in 5% and in 11%, respectively, of samples from children with trachoma. CONCLUSIONS: This new real-time PCR offers a sensitive, reproducible assay that produces results in <3 h. With panels of quantified Chlamydia species, this real-time PCR can be run with all real-time PCR equipment. Larger trials are needed to confirm the utility of this test in diagnosis and for therapeutic follow-up.

In vitro activity of midecamycin diacetate, a 16-membered macrolide, against Streptococcus pyogenes isolated in France, 1995-1999.

OBJECTIVE: To compare the in vitro activity of midecamycin diacetate to that of five other macrolides (erythromycin, clarithromycin, roxithromycin, azithromycin, and josamycin) and of clindamycin against 146 clinical isolates of Streptococcus pyogenes, with regard to three different phenotypes of erythromycin resistance. METHODS: Susceptibility pattern and resistance phenotype were determined by disk diffusion method and double disk test. Minimal inhibitory concentrations of antibiotics were obtained by the agar dilution method and evaluated according to the recommendations of the 'Comité de l'Antibiogramme de la Société Française de Microbiologie' (CA-SFM). The major determinants of erythromycin resistance in S. pyogenes (ermB, ermTR and mefA genes) were investigated by specific amplification protocols. RESULTS: Most of the isolates of S. pyogenes collected during 1995-99 were susceptible to midecamycin (93.8%), erythromycin (90.4%), clarithromycin (93.2%), roxithromycin (91.8%), azithromycin (88.4%), josamycin (94.5%), and clindamycin (94.5%). According to the CA-SFM criteria, 132 of the 146 isolates studied were susceptible to erythromycin (MICs < or = 1 mg/L), four were intermediate (MICs 2-4 mg/L), and 10 were resistant (MICs > 4 mg/L). Only nine isolates were midecamycin resistant (MICs > 4 mg/L), and the others were susceptible. The increased activity of midecamycin (MIC90 < or = 0.06 mg/L), as compared to erythromycin (MIC90 = 0.5 mg/L) and to other 14- or 15-membered macrolides, was related to the absence of the ermB determinant in seven isolates which displayed an efflux phenotype (five isolates) or an inducible resistance phenotype due to an ermTR determinant (two isolates). CONCLUSION: Midecamycin diacetate is active against most S. pyogenes strains isolated in France and may represent an attractive alternative to the treatment of streptococcal infections due to resistant isolates with efflux of erythromycin.