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Comparative studies of the efficiency of PCR overcoming isolation tests
by cultivation are already produced for over 20 years (Kessler
et al
., 1993;
Matsiota-Bernard
et al
., 1994; Ng
et al
., 1997).These studies showed that up to
36.3% of the samples identified by PCR were grown in non-selective medium
for
Legionella
sp. either for environmental, broncho-alveolar lavage (BAL),
aspirated intratracheal or urine collected from patients with pneumonia
samples.
On the other hand, concerns with the detection of
Legionella
by the PCR
techniques are discussed since 1997, when the presence of PCR inhibitors
in all samples was observed. To solve this problem a purification of the
genomic DNA prior to the reaction PCR or diluting material was proposed.
Furthermore, the use of negative and positive control during testing is crucial
for validation of any assay. The negative control provides confirmation that
the entire analytical process is free of error and contamination. Van der Zee
et al
. (2002a) detected the presence of
Legionella
contaminant in columns of
DNAextraction froma commercial kit, allowing the observation that external
contamination is possible, which could result some false positive result. Also,
the presence of the positive control allows to confirm the effectiveness of
each step of the protocol implemented, for example, the effectiveness of DNA
extraction, of the PCR reaction and the presence of inhibitors of the reaction.
For positive control, culture of
Legionella
can be used or, more safely, use the
lambda DNA with terminal sequences complementary to the used primers
(JOLY
et al
., 2006).
The advantages of the methods based on the PCR technique compared
to the culture-dependent methods include sensitivity, accuracy and rapid
assessment of contamination in different samples. However, it has been argued
that the main drawback of this strategy is the fact that it does not distinguish
