How molecular biology is changing the diagnosis of respiratory infections in critically ill patients Ricardo Serrano PhD. MD Department of Intensive Care Medicine General University Hospital of Alicante (Spain)
Inappropiate initial antibiotic therapy Excess morbidity and mortality Excessive antibiotic therapy Antibiotic resistance
Need for new microbiological diagnosis methods in respiratory infections TIME OF MICROBIOLOGICAL IDENTIFICATION TIME OF RESISTANCE PATTERN TIME TO CPIS 6 (VAP)
Microbiology VAP. Envin-Helics 2013 Source: ENVIN-HELICS report 2013
Am J Respr Crit Care 2003; 168: 173
Gram + Culture Serology Antigen detection VAP Microb Diagnosis MOLECULAR BIOLOGY
TRICORDER SCAN
Clinical aplications of molecular diagnosis Resistance Identification Molecular diagnosis VAP Quantification
Timothy F. Murphy Thorax 1990; 42: 452-459
Molecular Diagnosis in respiratory infections Real-time PCR FISH, FilmArrays Mass spectrometry
Angela M. Caliendo et al. CID 2013; 57 (Suppl 3): S139-S170
Single assay from unprocessed clinical Nucleic acid purification Reverse transcription PCR amplification Melting curve analysis sample
S. aureus identification Bacterial identification: MSSA and MRSA a) real-time PCR b) real-time PCR + hibridization probe VanA/VanB genes Time: 1-2 hours DNA targets: - specific genes - SSCmec - orf X region In reference to culture Sensibility: 75-85% Specificity: 94-96% Neg. Pred. Value: 99%
Hassan H, Shorman M. Int J Microbiology 2011
Cercenado E. J. Clin. Microbiol 2012; 50 (12): 4095-4097
Real-time PCR: HAP/VAP P. aeruginosa / A. baumanii Staphylococcus aureus / meca S. aureus / Panton-Valentine Leukocidin Kit PASM (Pseudomonas, Acinetobacter, Staphylococcus aureus, MecA)
Real-time PCR: specific situations CAP Flu outbreaks Inmunosupression Neumolysin. (Strep. pneumoniae) Mycoplasma / Chlamydia pneumoniae Legionella spp. / L. pneumophila Influenza virus A/B (H1N1,H1,H3) M. tuberculosis Complex/ R INH y RIF A. fumigatus, P. jiroveci Influenza virus A/B (H1N1,H1,H3) CMV, EBV, HSV 1and 2, VZV Infants Syncytyal respiratory virus (SRV)
Real-time PCR: RESISTANCE DETECTION Kit CTXE(ESBL): CTX-M groups 1, 2 and 9 Kit KPCK (CKP) Kit IMVI: MBL, IMP/VIM Kit AMPC: AmpC plasmidic betalactamase AnyPlex: Van A, Van B, Van C MagicPlex: Van A, Van B, Van C
Past Limitations Low cost Not especial trained perssonel Low risk contamination Easy interpretation of results Real-time PCR Increased cost Trained personnel Risk of contamination Lack of validation of some assays Complex interpretation of results Actual Strengths
MALDI-TOF (MS) (Matrix-Assisted Laser Desorption/Ionization- Time-Of-Flight)
MALDI-TOF (MS) (Matrix-Assisted Laser Desorption/Ionization- Time-Of-Flight) Protein analisis (mainly ribosomal proteins) Mass range 2.000 to 20.000 Da Microbial identification of cultured isolates Rapid identification method (minutes) Working in extend utilities: Bacterial identification from clinical samples Antibiotic resistance Typing different microorganisms Identification Fungal, mycobacterium, viruses
Wang Ye Ri et al. Biomed Environ Sci, 2013; 26 (6): 430-436
Yun Fong Ngeow et al. Sensors 2013,13, 15.242-15.251 Michelle Pignone et al. J Clin Microb 2006; 44 (6): 1963-1970 Chalupova et al. Biotecnology Advances 2013: in press
Reiner Schaumann et al. Med Sci Monit 2012 ; 18 (9): MT71-77
Johansson et al. BMC Microbiology 2014: 14-89
Johansson et al. BMC Microbiology 2014: 14-89
Antonella Mencacci et al. J Clin Microb 2013; 51 (2): 603-606
MALDI-TOF (MS): Limitations Low analytical sensibility utility (from growth colonies or sediment from blood culture) High cost of equipment and maintenance No cost/effective if < 5.000 identifications/year Need of positive/negative and calibration controls Regulatory issues. Difficulties to discriminate filogenetic similar microorganisms Difficult diagnosis of polimicrobial infections Low possibility of toxins identification Not authorities approval
Molecular diagnosis at General University Hospital of Alicante (Spain) QUANTITATIVE REAL TIME PCR : CMV, EBV QUALITATIVE REAL -TIME PCR: HSV 1 and 2, VZV, Influenza A (H1/N1)/ B, MSSA/MRSA, Bordetella pertusis, Mycobacterium tuberculosis MALDI-TOF MS: Bacteria/Fungal
MALDI-TOF MS in respiratory infection at GUHA (Spain) < 2 h: Gram stain from respiratory sample 24 h: MALDI-TOF identification 48 h: antibiogram C P I S