Rapid fungal identification in direct-positive culture- negative biopsies by PCR coupled to electrospray ionization mass spectrometry

Ref ID: 19357

Author:

A. Alanio, J. M enotti, S. Mercier-Delarue, A. Bergeron,
J. M. Molina, M. Legrand and S. Bretagne

Author address:

Hopital Saint Louis, APHP, Universit e Paris Diderot, France

Full conference title:

6th Trends in Medical Mycology 2013

Date: 11 October 2014

Abstract:

Objectives For invasive fungal diseases, results of direct examination
of biopsies provide the first clue for initiating antifungal therapy.
Therapeutic adjustment needs identification based on fungal culture,
which is unfortunately often negative (1). Moreover, direct examina-
tion alone cannot differentiate between very different molds such
hyaline filamentous fungi which can have different susceptibilities to
antifungal drugs. An identification as soon as the direct examination
is positive could lead to adapted antifungal treatment and will antici-
pate the risk of delayed or negative culture.
Methods We took advantage of PCR coupled to electrospray ioniza-
tion mass spectrometry (PCR/ESI-MS) which has been proposed for
fungi identification from respiratory specimens (2) or positive blood
cultures (3). Genus and species level assignment relies on (i) 16 single-
plex PCR assays using broad range primers which target nuclear or mi-
tochondial ribosomal RNA genes performed after standardized DNA
extraction, (ii) rapid determination of the amplicon’s base composition
using electrospray ionization mass spectrometry, and (iii) comparison
of the amplicons obtained to a specific database generated by the man-
ufacturer using reference strains and public databases. We tested here 4 direct-positive culture-negative biopsies from 3 patients using this
technique to obtain rapid fungal identification.
Results The molecular fungal identifications obtained were consis-
tent with the direct examination results (Table 1). The Qscore was
>0.96 for every sample. The Level criterion was >30 for all samples
but two for which it was just above 10 (broad assays for Patient 3).
Two seriate biopsies of the same lesion from Patient 2 gave identical
results showing the reproducibility of the method. For Patient 3,
multiple fungi were identified in accordance with direct examination
results (Table 1). For this specific patient, Aspergillus flavus, Purpureo-
cillium lilacinum or Rhizopus microsporus grew in culture from other
sampled sites with identification based on macroscopy, microscopy,
and ITS-sequencing excluding the risk of false positivity or miss-iden-
tification of the PCR/ESI-MS results.
Conclusion PCR/ESI-MS is of obvious interest for rapid and reliable
identification of fungi from biopsies. Indeed, identification can be pro-
vided in a working day in contrast with ITS sequencing. Another
advantage over ITS sequencing is to easily detect mixture of fungal
elements in the clinical samples.
(1) Arendrup Bone Marrow Transpl. 2012; (2) Simner JCM 2013;
(3) Kaleta JCM 2011

Abstract Number: o1.5

Conference Year: 2013

Link to conference website: NULL

New link: NULL


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