Culture Vs. qpcr. Daniel Grandio Gonzalez

Transcription

1 Culture Vs. qpcr Daniel Grandio Gonzalez

2 Introduction Legionelosis, Pontiac Fever, Mycobacteriosis Legionella: Hot water tanks, showers, cooling towers, swimming pools Environmental Mycobacteria: Purified water for hospital use. Points covered Easy approach to bacterial cycle Culture methods Vs. qpcr method. Pros and cons. Limitations. Suitability of each method. Result comparison Differences on taking samples

3 A Bacteria's life Physiological states of a cell: Alive Stationary phase Replicative phase Mature infectious VBNC (viable but not culturable) Dead Ecological states of a cell: Planktonic free cells Sessile cells (As part of biofilm) Hosted cells (intra-amoeba/ciliates)

4 Culture methods Culture: based on viable culturable bacterial cells. Cells in contact with a growth medium, where they replicate until they are visible as Colonies or MNP wells. Selective mediums special formulation to inhibit growth of undesirable microorganisms (i.e. use of antibiotics) Subculture and confirmation of presumptive colonies. Biochemical characteristics. Antibody agglutination Staining and microscopically observation Concentration Sample Culture Results are given in COLONY FORMING UNITS (cfu) Confirmation

5 Understanding Genetics DNA Nucleic sequence TACGATGC ATGCTACG Gene Amplification: Multiplication of nucleic sequences from a template chain. Primers: Artificial nucleic acid sequences that match with specific DNA sequence and serve as a guide for amplification. Fluorescent probe: Artificial nucleic sequence linked to a fluorescent molecule that matches with a specific DNA sequence revealing the amplification. Calibration Curve: Mathematical function that establishes the relationship between an amount of analyte present in a chemical reaction and the response of the measuring instrument.

6 qpcr method qpcr: amplification and labelling of distinctive DNA sequences with a set of specific primers+fluorescent probe. Initial concentration can be extrapolated from the calibration curve. Sample Concentration DNA extraction and purification qpcr Target DNA sequence + Appropriate primers + Fluorescent probe + Polymerase enzyme + Precise conditions = Amplification Fluorescent signal Results are given in GENOMIC UNITS (GU)

7 Culture Method: Pros vs Cons Gold Standard. Very well known and widely used. Cheap and easy to perform. Isolation of living cells useful for further study. Highly validated and normalized. Slow and time consuming (7-12d Legionella; 28-42d Mycobacteria). Methods developed for specific species and strains. A few environmental mutant strains may not grow. Non mature and stressed cells (VBNC) can be undetectable. Background flora competence Growth inhibition. Result interpretation relies on analysts experience and skills False negatives or underestimations can occur.

8 qpcr. Pros vs Cons Well developed technology (late 80 s). Fast (<24 hours). High specificity. High sensitivity. High negative predictive value. Objective and simple interpretation of results. Detection of all available cells (alive, dead, VBNC ). Not recognised as a normal procedure for routine analysis by regulatory organisms. Need of normalization improvements. Inhibitors present in the matrix. High level training requirements + Expensive reagents = High priced. Detection of dead cells.

9 How qpcr is overcoming its limitations High level training requirements + Expensive reagents = High Priced Increase of diagnostic PCR use Market competition Low prices Evolution of sample preparation systems. Commercial kits Not recognised as a normal procedure for routine analysis + Need of normalization improvements. Validation documents and standard guides (ISO/TS 12869:2012) Standardization of gene targets Intercollaborative trials. Inhibitors present in the matrix. Evolution of sample preparation systems. Dilution of samples. Detection of dead cells. Intercalating agents (PMA) block free DNA and DNA from dead cells. qpcr rapid screening + culture of positive samples Target molecule. mrna instead of DNA (RT-PCR, NASBA).

10 Quantitative result interpretation and comparison Target Detection technique Result cfu GU Different targets Different techniques Different results Main causes of BIG disagreement Detection of dead cells (i.e. after disinfection) increases the rate GU/cfu. Inefficient disinfection (wrong biocides or concentrations) Presence of biofilm (rich in VBNC) Ideal solution: 1. Long term combined monitoring 2. Understanding of system s microbiota dynamics 3. Periodical qpcr monitoring

11 Qualitative result interpretation and comparison qpcr Positive Culture Positive Sample is positive Culture Negative Cells are present but may be dead, VBNC or culturable but below the limit of detection. qpcr Negative There are several possible causes: Inhibition of qpcr by sample matrix. Variation on Limits of Detection (dilution, not enough sample ) Sample is negative

12 What to use qpcr and Culture for Regulatory compliance: Regulated culture methods. qpcr as a predictive tool. Outbreak: qpcr allows a large number of samples (i.e. every outlet in a large building) to be tested quickly. Targeted remediation in the areas needed. Culture for positive confirmation Routine monitoring: A combination of both methods, doing culture in the case of a positive qpcr qpcr also useful to detect Legionella/mycobacteria present at too low level or in VBNC state. Early warning qpcr not useful after disinfection. PMA-qPCR will be useful.

13 Recommendations for sampling Mycobacteria culture typically requires 2x100mL depending on the application. At least 250mL should be sampled in a normal microbiological sample bottle. Legionella culture requires between mL depending on which concentration method is used. Ideally mL should be taken in a standard microbiological sample bottle. qpcr usually requires 1L, smaller volumes will adversely affect the quantification limit of the method. Samples should be taken in a standard microbiological sample bottle.

14 Summarizing Time Price Cells detected Measuring Units Regulation Routine monitoring Outbreak Disinfection Regulatory compliance qpcr <24hr Moder ated All GU/vol Low Yes Yes Pre Not currently. Soon PMA-qPCR <24hr High All but dead GU/vol Low Yes Yes Pre-post Not currently. Soon Legionella culture 7-12d Low Only viable and culturable cfu/vol High Yes Not effective Pre-post, late response Yes Mycobacteria culture 28-42d Low Only viable and culturable cfu/vol High Yes Not effective Pre-post, late response Yes

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