ࡱ> molg"\Qbjbjhh".R|\|\'-NN ,^0$$$$$]]]]]]]$_bJ]]$$`^]]]]]]A$$]]]]]]]]]]$ sɓW]]]v^0^]]b\jb]]b]]$]]]]]]^bNP :"=AB8BCB <>;5:C;O@=>W 1V>;>3VW V 35=5B8:8  #:@0W=8 # !  14 ;8AB>?040 >1 11:00 :>=D5@5=F  70;0  # (49 ?>25@E) 0AB>AC20==O <>;5:C;O@=>W 1V>;>3VW 4> 4V03=>AB8:8 V=D5:FV9=8E 70E2>@N20=L (28O2;5==O 10:B5@V9, 2V@CAV2 B0 2V;L=>682CG8E 0<51) 5:FVO @. 01;> >;L4H<V4B0, ;V=V:0 "Des Quinze-Vingts" (0@86, $@0=FVO) ____________________________________________________________________________ - . Publicaciones del area de la Microbiologia no limitadas a la virologia I-a Effects of topical anaesthetics and fluorescein on the real-time PCR used for the diagnosis of Herpesviruses and Acanthamoeba keratitis Goldschmidt P, Rostane H, Saint-Jean C, et al British Journal of Ophthalmology 2006;90:1354-1356. 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. I-bNew test for the diagnosis of bacterial endophthalmitis British Journal of Ophthalmology 2009;93:1089-1095. P Goldschmidt, S Degorge, D Benallaoua, E Basli, L Batellier, S Boutboul, C Allouch, V Borderie, L Laroche, C Chaumeil Background: Diagnosis of bacterial endophthalmitis (BE) often fails due to: (1) insufficient volumes of vitreous fluid (VF) and aqueous humour (AH); (2) lack of sensitivity of culture; (3) antibiotic treatments; (4) polymerase chain reaction (PCR) cross-contamination; and (5) limitations on the interpretation of the real-time PCR melting curve. We developed a fast real-time (f-real-t) PCR to improve the performance of the laboratory diagnosis of BE. Methods: The following samples were processed after adding an internal control: phosphate buffered saline (PBS); VF, AH and cell suspensions spiked with Bacteria (Bac); VF and AH from patients with endophthalmitis; and VF and AH from non-infective patients. DNA was extracted (MagNA Pure) and added to four tubes containing selected primers and probes for the identification and quantification of all Bac and eight genera by f-real-t PCR. Diagnostic performances based on direct microscopic examination, culture and f-real-t PCR were compared. Results: The f-real-t PCR detected at least 0.01 colony-forming units (CFU) of Bac/l with no cross-reactivity with fungi. Correlation with culture-positive results was 100%. Sixty per cent of BE samples tested culture-positive, but f-real-t PCR tested positive for 90%. Samples from non-infective cases were negative. Conclusion: The f-real-t PCR detected and quantified Bac, Staphylococci, Streptococci, Haemophilus, Pseudomonas, Enterobacteria, Acinetobacter, Propionibacteriacae and Corynebacteria in one run. Cultures required several hours to days (with a non-negligible number of false-negative results) and the f-real-t PCR was completed in 90 min. The f-real-t PCR is presented as a new tool for the diagnosis of BE: its usefulness requires validation with larger series of samples. I-c Reducing HIV Mortality: A New Paradox for Practitioners Working in Countries with Socialized Health-care Systems. Goldschmidt P. Trop Med Health. 2011 Jun;39(2):59-62. The milestones marking substantial changes in the lives or in the survival of humans deserve to be remembered. It has been only 11 years since we experienced an event that not even the most optimistic amongst us would have predicted before 1997. Let us place the facts in time. At the beginning of the 80s, we faced the distressing reality that more than three quarters of all children, men and women found to have antibodies directed against a new infectious agent named human immune deficient virus (HIV) were bound to die. The mere reactivity of the serum of a human being against a virus characterized in 1983 (antibodies) handed an almost inevitable sentence of death. At that time the evolution of this viral infection was assessed by the quantification of a sub type of white cells, the auxiliary lymphocytes or CD4. This count was the principal evidence for most of the predictions on how a person might survive without degradation, and the value of such cells was the abacus used to forecast the time when an individual would develop irreversible blindness, to anticipate respiratory failure, and to predict the time before weakness would appear after devastating diarrhea, etc. We should recall that the CD4 cell count was even used as a predictor of the initiation of cognitive shrinkage, forecasting dementia as well as the signs that would take hold of personality as a consequence of infections or neoplastic transformations in the encephalitic mass. II-Biologia Molecular aplicada a enfermedades infecciosas II-a Enzymatic treatment of specimens before DNA extraction directly influences molecular detection of infectious agents. Goldschmidt P, Degorge S, Merabet L, Chaumeil C. PLoS One. 2014 Jun 17;9(6):e94886 Enzymatic treatment of specimens before DNA extraction directly influences molecular detection of infectious agents. Goldschmidt P(1), Degorge S(1), Merabet L(1), Chaumeil C(1). PLoS One. 2014 Jun 17;9(6 INTRODUCTION: Biological samples, pharmaceuticals or food contain proteins, lipids, polymers, ammoniums and macromolecules that alter the detection of infectious agents by DNA amplification techniques (PCR). Moreover the targeted DNA has to be released from the complex cell walls and the compact nucleoprotein matrixes and cleared from potential inhibitors. The goal of the present work was to assess the efficiency of enzymatic pretreatments on infectious agents to make DNA available for further extraction and amplification. METHODS: Staphylococcus epidermidis, Streptococcus mitis, Propionibacterium acnes, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Fusarium solani were mixed with an internal control virus and treated with: 1) proteinase K; 2) lyticase and 3) lyticase followed by proteinase K. DNAs was manually extracted using the QIAmp DNA Mini kit or the MagNA Pure Compact automate. DNA extraction yields and the inhibitors were assessed with a phocid Herpesvirus. Bacterial detection was performed using TaqMan real-time PCR and yeasts and filamentous Fungi with HRM (real-time PCR followed by high-resolution melting analysis). RESULTS: Viral DNA was released, extracted and detected using manual and automatic methods without pre enzymatic treatments. Either the manual or the automatic DNA extraction systems did not meet the sensitivity expectations if enzymatic treatments were not performed before: lyticase for Fungi and Proteinase K for Bacteria. The addition of lyticase and proteinase K did not improve results. For Fungi the detection after lyticase was higher than for Proteinase K, for which melting analysis did not allow fungal specification. DISCUSSION: Columns and magnetic beads allowed collecting DNA and separate PCR inhibitors. Detection rates cannot be related to DNA-avidity of beads or to elution but to the lack of proteolysis. II b - Rapid detection and simultaneous molecular profile characterization of Acanthamoeba infections. Goldschmidt P, Degorge S, Benallaoua D, Batellier L, Di Cave D, Chaumeil C. Diagn Microbiol Infect Dis. 2012 Oct;74(2):137-41. Rapid detection and simultaneous molecular profile characterization of Acanthamoeba infections. Diagnosis of Acanthamoeba by microscopic examination, culture, and polymerase chain reactions (PCRs) has several limitations (sensitivity, specificity, lack of detection of several strains, cost of testing for discrimination among strains). We developed a new high-resolution melting real-time PCR (HRM) to detect and characterize Acanthamoeba infections. HRM performances were evaluated with strains from the American Type Culture Collection (ATCC) and with 20 corneal scrapings. The DNA extracted from specimens were amplified, detected, and characterized in 1 run using 2 original primers diluted in a solution containing an intercalating dye. Detection and molecular characterization of Acanthamoeba infections could be achieved in less than 2.5 h with a dramatic reduction in cost of reactants (postamplification procedures and radioactive or fluorescent-labeled molecular probes were unnecessary). HRM detection limits were 0.1 cyst/L or less (including genotypes T5 and T11), and its sensitivity and specificity were higher than other molecular tests. For the tested strains from the ATCC, the HRM drafted 4 different profiles: Type I (genotypes T2 and T4), Type II (T5 and T7), Type III (T8), and Type IV (T1, T3, T6, T9, T11, T12, and T13). II c- Detection by broad range real time PCR assay of Chlamydia species infecting human and animals P Goldschmidt, H Rostane, M Sow, A Gopogui, L Batellier, and C Chaumeil Br J Ophthalmol. 2006 Nov; 90(11): 1425 1429. Background: Tests available for molecular diagnosis of chlamydial infel $ & ( 4 b h ^""33446DFFFFHHIIII K KKKMMNPPP`PbPRQTQZQ\QŽh%UhkOJQJ^Jheyhk6heyhkhk5 hk5hk5CJaJhkhk5CJaJ hkhkhkhkhk5;.l  & ( f ^ [ E1" Dy$a$gdey`gdkgdk$a$gdkGg;/|j [ O!!!B"\"^"""#$a$gdeygdk#E#g##$3$$$%k%%&G&&&2'''!(o((()j)) *T***4+gdk4+++++,_,,,@---1.|../e// 00T112@3333345gdk5t5$ElF:IL\Qgdkctions 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/l). 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. ,1h. 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