LABORATORIO DE ANÁLISIS: Impacto de las técnicas rápidas en las rutinas de trabajo en laboratorio.
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Dr. Jorge Álvarez – LABORATORIO DE ANÁLISIS: Impacto de las técnicas rápidas en las rutinas de trabajo en laboratorio. Ponencia presentada durante el Primer Congreso Internacional de Detección Rápida de Legionella celebrado el 26 de Noviembre de 2015 en la Universidad Jaime I de Castellón (www.ilfdcongress.com).
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LABORATORIO DE ANÁLISIS: Impacto de las técnicas rápidas en las rutinas de trabajo en laboratorio.
- 1. Rapid ID techniques Impact of rapid testing on the routine work at a Laboratory RESTRICTEDCONFIDENTIAL PUBLICINTERNAL
- 2. Latis Scientific Ltd UK Labaqua Spain Aqualogy Suez Environment RESTRICTEDCONFIDENTIAL PUBLICINTERNAL
- 3. Waterborne pathogens Sampling Transport Report and Corrective actions Laboratory analysis Major health risk Waterborne pathogens Wide dispersion Quality assessment workflow Representative Stability TimeStress RESTRICTEDCONFIDENTIAL PUBLICINTERNAL
- 4. Gold standards Strong regulation and legislation. Used for centuries. Limits and threshold normalised. Cheap and easy to perform.Laboratory analysis Concentration Culture Confirmation Legionella 7-10 daysMycobacteria 28 days Pseudomonas 2-3 Days DRAWBACKS • Poor selectivity and specificity • Low recovery rate • Time consuming • Long time lead RESTRICTEDCONFIDENTIAL PUBLICINTERNAL
- 5. Rapid ID techniques Rapid ID methods: Results in less than 24 hours. Consolidated technologies in research area. - Biochemistry assays - Based on biochemical reactions. Bacterial enzymes catalyse different substrates to reveal a change of colour. - Nucleid acids methods - Based on a specific sequence of DNA. It normally involves an amplification step. Detection by fluorescence. - Immuno related assays. - Use of specific Antibodies to mark or isolated the target RESTRICTEDCONFIDENTIAL PUBLICINTERNAL
- 6. Biochemistry assays • Results in 24 hours • Based in biochemical reactions combined with selective growth culture. (e.g. B-galactosidase) • Viable and cultivable cells DRAWBACKS • Results expressed as most probable number (MPN). • Few species available. • Limit of detection similar to culture. • Expensive • API strips for Biochemical identification. • Pure cultures • Low discrimination power for environmental strains. • Ambiguity BIOLUMIX RESTRICTEDCONFIDENTIAL PUBLICINTERNAL
- 7. DNA techniques qPCR • Results in less than 6 hours. • Specific set of primers and probe allows to great specificity and sensitivity. • Dead + Viable cells (including VNCB) • EMA/PMA treatment Viability qPCR – Viable cells. • Few commercial kits. • New regulations Others • Microarrays • FISH • Isothermal amplification DRAWBACKS • Expensive • High level training requirements • Inhibitors • Lack of normalization RESTRICTEDCONFIDENTIAL PUBLICINTERNAL
- 8. Immune-related assays Results within hours. Detection of Viable cells. Automatization Flow cytometry Immunomagnetic bead isolation and absorbance (Legipid) DRAWBACKS • Matrix effect • Expensive equipment • High qualified technicians • Difficult interpretation of results • Specific markers (Ab) hard to find DRAWBACKS • Matrix effect • Medium specificity • Medium limit of detection (Improves culture) RESTRICTEDCONFIDENTIAL PUBLICINTERNAL
- 9. Rapid ID techniques Advantages/Drawbacks: qPCR Legiped Flow cytometry Lead time 5-6 hours 1-3 hours 1-3 hours LOD Low Medium-Low Low Viable cells PMA (Limited) Yes Yes Automatization Yes Yes Yes Technical training Intermediate Easy High Sensitivity/Precision High/High High/Medium High/Medium RESTRICTEDCONFIDENTIAL PUBLICINTERNAL
- 10. Real case study Legionella Level Culture Legiped qPCR Not detected 10 5 5 Low 0 5 5 Medium 5 5 5 High 5 5 5 Sample L spp L pn Sg1L pn SG2-14 L.spp GU/L cfu/L 543509 ND ND >15000 376000 3693 543510 ND ND 200 464000 1451 543511 ND ND 100 204000 2070 543512 ND ND ND 48800 711 543513 ND ND 100 436000 1565 543514 ND ND 850 402000 2980 543515 ND ND ND 63600 711 543516 ND ND 150 246000 1936 543517 ND ND ND 90800 1238 559509 ND ND 150 196600 1396 559511 ND ND 300 318000 2502 559200 ND ND ND 96400 2656 559202 ND ND ND 1833 212 559203 ND ND ND 2753 212 559204 ND ND ND 28800 1044 559205 ND ND ND 24200 ND 20 controlled samples: • 5 not contaminated • 5 low level contamination • 5 medium level contamination • 5 high level contamination 53 real samples Culture qPCR Legipid RESTRICTEDCONFIDENTIAL PUBLICINTERNAL
- 12. Real samples qPCR(GU) Legipid 24200 ND 57500 ND 56500 ND 142000 ND Methods are not comparable: • Dead cells • Matrix Effect • Different sensitivity RESTRICTEDCONFIDENTIAL PUBLICINTERNAL
- 13. Conditions: Fast Sensitive Specific Precise Detection of all Viable cells. Easy to perfom Automatization Portable or in-situ Monitoring online Future methods RESTRICTEDCONFIDENTIAL PUBLICINTERNAL
- 14. Rapid ID techniques RESTRICTEDCONFIDENTIAL PUBLICINTERNAL
- 15. Conclusions Culture method is obsolete • Poor sensitivity, specificity and low recovery (high LOD) qPCR and Legipid are not comparable. • Different results lead to different corrective actions • Both have good technical parameters (LOD, sensitivity) • qPCR detects also dead cells (after disinfection) • Matrix effects Currently, no alternative method can overcome limitations by itself Which one to trust RESTRICTEDCONFIDENTIAL PUBLICINTERNAL
- 16. Sampling Transport Report and Corrective actions Laboratory analysis Real time In-situ Monitoring Data online Control centre: Quality assesment Inmmediate response Corrective actions RESTRICTEDCONFIDENTIAL PUBLICINTERNAL
- 17. Rapid ID techniques RESTRICTEDCONFIDENTIAL PUBLICINTERNAL