Can panfungal PCR detect and identify fungal species in culture negative samples from patients suspected of invasive fungal infection?

Ref ID: 19390

Author:

M. Lengerova, D. Palousova, P. Bejdak, I. Kocmanova and
Z. Racil

Author address:

Masaryk University and University Hospital Brno, Czech Republic

Full conference title:

6th Trends in Medical Mycology 2013

Date: 11 October 2014

Abstract:

Background Apart from cases of invasive aspergillosis and invasive
mucormycosis, are also increasingly common infections caused by the
so called “œrare fungi”. If culture of clinical material is negative, repre-
sents PCR and subsequent sequencing, the only possibility to deter-
mine the etiological agent. For this application seems to be ideal
panfungal PCR, in which are the primers directed to conserved regions
fungal genome. Identification of species is done by sequencing. Panfun-
gal PCR is very suitable for fast and accurate identification of cultures
of morphologically similar species of yeasts and filamentous fungi. The
great advantage of PCR over culture is little chance of influencing the
outcome as a result of the initiation of empirical antifungal therapy,
because PCR is able to detect DNA of non-viable organisms. On the
other hand, we face the difficulty of interpretation of positive detection
of fungal pathogens in non-sterile clinical samples (e.g. bronchoalveo-
lar lavage, sputum, samples from autopsy).
Material and Methods In our study, we tested various clinical sam-
ples using two independent panfungal assays (one-round PCR and
nested PCR) bordering the ITS rDNA gene. Fungal species was deter-
mined by sequencing of PCR products. Since we often got mixed
sequences by direct sequencing of PCR products, we decided to clone
PCR products first. After transformation were colonies screened by
panfungal primers, analyzed on an agarose gel and PCR products of dif-
ferent length were selected for sequencing. Overall, between 2009-
2012 we tested samples from 108 patients (79 BAL, 19 tissues). Indica-
tion for the examination of clinical samples using panfungal PCR was
mostly positive result of other diagnostic methods (histology, micros-
copy, galactomannan or 1,3-beta-D-glucan) and negative culture.
Results In our cohort of patients, we were able to identify several
rare pathogens as causative agents of invasive infections – e.g. Asper-
gillus terreus, Rhizomucor pusillus, Cladophialophora bantiana, Neosar-
torya pseudofisheri, Fusarium proliferatum, Schizophyllum commune.
Most of them were later confirmed by culture. The best results are
achieved when we were testing tissue samples (either fresh or fixed)
with positive histology. Interpretation of results of testing of BAL
samples was difficult due to frequent positivity of several fungal spe-
cies including those that are mostly considered to be contaminants.
Conclusions Panfungal PCR proved to be an excellent tool for precise
identification of rare fungi in fungal cultures. We were also able deter-
mine the causative species in histologically positive tissues. Due to dif-
ficult interpretation of the positive results has panfungal PCR only
limited use for testing of non-sterile clinical material (BAL, sputum).
This work was supported by: Supported by MH CZ – DRO (FNBr,
65269705).

Abstract Number: p073

Conference Year: 2013

Link to conference website: NULL

New link: NULL

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