Promises and pitfalls of diagnostic PCR

Ref ID: 19351

Author:

S. Bretagne and W. J. G. Melchers

Author address:

Pasteur Institute, Paris, France and Department of Medical
Microbiology, Radboud University Nijmegen Medical Centre,
the Netherlands

Full conference title:

6th Trends in Medical Mycology 2013

Date: 11 October 2014

Abstract:

In mycology, PCR data are still not included in the consensual crite-
ria for definitions of invasive fungal diseases although many PCR
assays have been developed over the previous 20 years. This is
mainly due to the critical balance of the technical versus the clinical
sensitivity in the molecular diagnosis of invasive fungal infections.
The generally very low amount of fungal DNA in clinical specimens
challenges the limits of PCR (technical sensitivity). The low amounts
of fungal copies also compromises the value of a positive PCR (clini-
cal sensitivity) as contamination by either PCR artifacts or environ-
mental DNA present as spores or contaminated consumables would
lead to false positive results. The quantitative PCR (qPCR) format is
now compulsory to decrease the risk of false positivity. On the other
hand, the slighter decrease of PCR yield will lead to false negative
results, hence the mandatory use of internal control, which can only
be optimized with the use of the qPCR format. Consensual procedures
based on the ’Minimum Information for the publication of real-time
Quantitative PCR Experiments’ (MIQE) guidelines can be now fol-
lowed to implement a PCR test in routine diagnostics.
However, the prevention of false positives cannot be technically
avoided when contamination relies on contamination in the sampling
tubes, in enzyme used for the reactions, or in infusions administrated
to the patient. This may be partially controlled by a careful design of
the primers to avoid “œpanfungal”ones able to amplify environmental
fungal DNA. The more specific, the lower the risk of false positives with
environmental DNA. However, also the more specific, the more likely
to miss rare or emergent species infection. Mismatches with human
DNA is also of concern, since the objective is to detect minutes of fun-
gal DNA among huge amounts of undesirable human DNA.
To propose a more defined procedure, we are also limited by the
still unsolved issue of the fungal DNA origin in the clinical speci-
mens. To start with blood, serum or bronchoalveolar lavage (BAL)
fluids rise different issues on how to extract the fungal DNA and how
to eliminate the putative PCR amplification inhibitors. That is why
the current trend is to favor serum or plasma over blood for the ease
of pre-analytical steps.
Once the technical issues are solved, remains the difficult point of
the clinical significance of a PCR-positive result (clinical sensitivity).
This seems simple when Aspergillus DNA is detected in CSF or serum.
It is obviously different in BAL since the interpretation can be infec-
tion, colonization, or by-standing presence in inhaled air. Similarly,
during candidemia episodes, Candida DNA is more often detected
when patients are already given antifungal drugs, suggesting that the
yeasts are no longer vital. Then, the clinical relevance of a PCR test
hugely depends on the time point of disease progression. The outcome
of a PCR test is often correlated to the at-risk period of an immuno-
compromised patient. The major significance of a PCR test would be
before the patient is treated with antifungal drugs. Moreover, to
include a PCR test in a screening strategy, the test must be evaluated
in clinical studies as the additional value of the PCR test compared
with the already clinically validated tests must be evaluated.
The comments discussed mainly focused on Aspergillus and Candida
infections. For some other common pathogenic fungi such as Pneumocystis
, the technical sensitivity is of lower concern since Pneumocystis
DNA in BAL is often present in huge quantities. Indeed,
Pneumocystis qPCR assays already favorably replace classical diagnos-
tic methods. The issue here is also the interpretation of a positive sig-
nal corresponding to a low fungal load.
In conclusion, the MIQE procedure brought a strong rationale for
the technical and clinical validation of qPCR assays. The primer
choice is still controversial and balances between very species specific
vs. pan-fungal strategy. The DNA extraction step must be adapted to
every clinical. A definitive acceptance should be achieved through
well-designed prospective studies.

Abstract Number: m13

Conference Year: 2013

Link to conference website: NULL

New link: NULL

Conference abstracts, posters & presentations

Showing 10 posts of 17325 posts found.

  • Title

    Author

    Year

    Number

    Poster