Multicentre study on the reproducibility of MALDI-TOF MS for nontuberculous mycobacteria identification

The ability of MALDI-TOF for the identification of nontuberculous mycobacteria (NTM) has improved recently thanks to updated databases and optimized protein extraction procedures. Few multicentre studies on the reproducibility of MALDI-TOF have been performed so far, none on mycobacteria. The aim of this study was to evaluate the reproducibility of MALDI-TOF for the identification of NTM in 15 laboratories in 9 European countries. A total of 98 NTM clinical isolates were grown on Lowenstein-Jensen. Biomass was collected in tubes with water and ethanol, anonymized and sent out to the 15 participating laboratories. Isolates were identified using MALDI Biotyper (Bruker Daltonics). Up to 1330 MALDI-TOF identifications were collected in the study. A score >= 1.6 was obtained for 100% of isolates in 5 laboratories (68.2-98.6% in the other). Species-level identification provided by MALDI-TOF was 100% correct in 8 centres and 100% correct to complex-level in 12 laboratories. In most cases, the misidentifications obtained were associated with closely related species. The variability observed for a few isolates could be due to variations in the protein extraction procedure or to MALDI-TOF system status in each centre. In conclusion, MALDI-TOF showed to be a highly reproducible method and suitable for its implementation for NTM identification

"Beware man's best friend".

International audienceCase report. To illustrate the usefulness of broad-range bacterial polymerase-chain-reaction (PCR) testing in osteoarticular infections by Capnocytophaga canimorsus. C. canimorsus is a gram-negative bacillus that was first identified in 1976. It is a normal inhabitant of the oral mucosa of dogs (26%) and cats (15%). The main clinical patterns are septicemia, which may cause fatal septic shock (in 30% of cases) but arthritis and discitis are possible. C. canimorsus is susceptible to many antimicrobials including β-lactam antibiotics. About a case report of 54-year-old man transferred to our institution for discitis, we discuss about PCR for C. canimorsus discitis diagnosis. At admission, the only abnormal physical finding was global pain and stiffness of the lumbar spine. Radiographs of the lumbar spine and pelvis showed lumbar spondylosis, degenerative disc disease, and previously known degenerative facet joint disease. Magnetic resonance imaging indicated L3-L4 discitis with damage to both vertebrae and adjacent discs. Findings were negative from blood and urine cultures, serological tests for the HIV and brucellosis, and sputum tests for Mycobacterium tuberculosis. A percutaneous biopsy of the L3-L4 disc was performed but the bacteriological studies recovered no organisms. A second needle biopsy was performed for broad-range 16S rDNA PCR testing, which identified C. canimorsus. Broad-range PCR provided the microbiological diagnosis of culture-negative discitis in our patient. Broad-range PCR should be considered in patients with culture-negative osteoarticular infections

"Beware man's best friend".

International audienceCase report. To illustrate the usefulness of broad-range bacterial polymerase-chain-reaction (PCR) testing in osteoarticular infections by Capnocytophaga canimorsus. C. canimorsus is a gram-negative bacillus that was first identified in 1976. It is a normal inhabitant of the oral mucosa of dogs (26%) and cats (15%). The main clinical patterns are septicemia, which may cause fatal septic shock (in 30% of cases) but arthritis and discitis are possible. C. canimorsus is susceptible to many antimicrobials including β-lactam antibiotics. About a case report of 54-year-old man transferred to our institution for discitis, we discuss about PCR for C. canimorsus discitis diagnosis. At admission, the only abnormal physical finding was global pain and stiffness of the lumbar spine. Radiographs of the lumbar spine and pelvis showed lumbar spondylosis, degenerative disc disease, and previously known degenerative facet joint disease. Magnetic resonance imaging indicated L3-L4 discitis with damage to both vertebrae and adjacent discs. Findings were negative from blood and urine cultures, serological tests for the HIV and brucellosis, and sputum tests for Mycobacterium tuberculosis. A percutaneous biopsy of the L3-L4 disc was performed but the bacteriological studies recovered no organisms. A second needle biopsy was performed for broad-range 16S rDNA PCR testing, which identified C. canimorsus. Broad-range PCR provided the microbiological diagnosis of culture-negative discitis in our patient. Broad-range PCR should be considered in patients with culture-negative osteoarticular infections

Multicentre prospective evaluation of histological and molecular criterion for diagnosis of prosthetic-joint infection

International audienceObjectives:This multicenter prospective study was performed to assess the contribution of broad range PCR diagnosis in prosthetic-joint infection (PJI).Methods:Adult patients treated for PJI at 7 centers were included between December 2010 and March 2012. Six per-operative samples were obtained for each patient, 5 for conventional cultures and 16S rRNA gene real-time PCR followed by sequencing, and 1 for histopathological classification according to Morawietz. Cultures and PCR were performed in a highly standardized manner, with 3 quality controls of PCR analyses. An infection was considered as proved (3 criteria: per-operative, bacteriological and histological), probable (clinical or bacteriological criterium), or excluded (no criterium). Molecular criterium for predicting PJI was determined using the bacteriological one as reference (&gt;=1 positive sample for virulent organism, and &gt;=3 positive samples for coagulase-negative staphylococci (CoNS) and P. acnes).Results:299 patients were included, 264 with suspicion of sepsis (S) and 35 as controls (C). The 264 S presented with acute (19%), or chronic suspicion of PJI (81%). Infection was proved or probable in 212/264 S (80%), with the bacteriological criterium in 189/212 S (89%). Out of these, 156 (83%) had monomicrobial and 33 (17%) polymicrobial infections. The isolated pathogens were S. aureus (40%), CoNS (25%), streptococci (14%), Gram-Negative rods (10%), and anaerobes 8%.Histology results were not available for 55 patients, leaving 244 patients available for analysis. Histological findings of infection (Morawietz types II or III) were present in 128/169 (76%) proved or probable infections, in 3 patients without any other criterium, and were absent in excluded infections (n=42) and controls (n=29). PCR results were not analysable for 32 patients (S=28, C=4), leaving 267 patients (S=236, C=31) available for analysis. Molecular criterium of infection was present in 63/68 (93%) proved infections, 83/124 (67%) probable infections, 3/42 excluded infections, 0/2 histological criterium alone and 2/31 controls. Molecular criterium of infection was absent in 34/189 (18%) culture-positive S, and present in 8/23 culture-negative S (8 patients treated with antibiotics).Conclusions:According to this multicenter prospective study, 16S rRNA gene real-time PCR is less susceptible than culture for diagnosis of PJI. Molecular analysis could be recommended in culture-negative patients who were receiving antibiotics.</p

Multicentre external quality control evaluating universal 16S polymerase chain reaction (PCR) in the diagnosis of bone and joint infections

International audienceObjectives:During a multicentrer French Study performed to assess the contribution of 16S PCR in the diagnosis of prosthesis osteoarticular infections, 300 patients were included from December 2009 to April 2012. An external quality control (QC) was considered essential due to the diversity of molecular equipment for each laboratory. Three sets were held, for each including 4 bacterial DNA extracts (E) and 4 crushed osteoarticular deep samples.Methods:Extraction: 0,2 ml of pretreated S (PK, 37 °C, 3h) with elution in 0.1 ml. Four laboratories used Qiagen manual extraction and 3 others used automated extraction 1 MagnaPur Roche, 1 Easy Mag, BioMérieux and 1 iPrep, Invitrogen. Real time 16S PCR with SybrGreen was performed with degenerate primers amplifying 658pb followed by sequencing. In the 7 centers, PCR thermocyclers used were 2 MX 3000p Agilent, 1 Roche Light Cycler, 1 Abi 7900 and 1 Applied Step one plus, 1 Smartcycler Cepheid,1 Biorad Chrono 4 and for PCR premix, Takara premix exTaq, Applied, Promega and Biorad were used.Results: 168 QC were sent and 160 responses were analyzed (1 laboratory did not participate in the first QC series). Expected results were obtained in 97.5% for Extracts and 95% for Samples. Sensitivity and Specificity were 100 and 90% for E and 93.3 and 100% for S. Ct standard deviations (SD) for E were from 1 to 9 while SD was 2 to 7 for S. For centers using the same premix, the results were closer, SD 0.5 to 1.5 (3 Ct gap max). For S, no influence of extraction system was observed.Conclusion:If extraction system had no influence, premix seems to be the most important factor influencing the value of threshold. This QC demonstrates the possibility to obtain good and homogeneous results by using the same 16S PCR in laboratories with different equipments for molecular bone and joint infection diagnosis

Evaluation of 16S rRNA gene PCR sensitivity and specificity for diagnosis of prosthetic joint infection: a prospective multicenter cross-sectional study.

International audienceThere is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥ 1 positive sample for strict pathogens and ≥ 2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis