Culture Vs. qPCR

Daniel Grandio Gonzalez

Introduction

Legionelosis, Pontiac Fever, Mycobacteriosis

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

Legionella: Hot water tanks,

showers, cooling towers,

swimming pools

Environmental Mycobacteria:

Purified water for hospital use.

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)

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 observationCulture

Confirmation

Concentration

Sample

Results are given in COLONY FORMING UNITS (cfu)

Understanding Genetics

• 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.

TACGATGC

ATGCTACG

Gene

DNA Nucleic sequence

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.

Results are given in GENOMIC UNITS (GU)

Target DNA sequence + Appropriate primers + Fluorescent probe + Polymerase

enzyme + Precise conditions = Amplification Fluorescent signal

DNA extraction and purification

Concentration

qPCR

Sample

qPCR method

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.

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.

• 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).

How qPCR is overcoming its limitations

Quantitative result interpretation and comparison

cfu

≠

GU

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)

Target Detection technique Result

Ideal solution:

1. Long term combined monitoring

2. Understanding of system’s microbiota

dynamics

3. Periodical qPCR monitoring

Different targets Different techniques Different results

Qualitative result interpretation and comparison

qPCR Positive

Culture Positive Culture Negative

Sample is positive

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

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.

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 200 – 1000mL depending on which

concentration method is used. Ideally 500 – 1000mL 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.

Summarizing

Time PriceCells

detected

Measuring

UnitsRegulation

Routine

monitoringOutbreak Disinfection

Regulatory

compliance

qPCR <24hrModer

atedAll 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

culture7-12d Low

Only viable

and culturable cfu/vol High YesNot

effective

Pre-post, late

responseYes

Mycobacteria

culture28-42d Low

Only viable

and culturablecfu/vol High Yes

Not

effective

Pre-post, late

responseYes

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