BACKGROUND Fluorescence
in situ hybridisation (FISH) associated with Tyramide Signal Amplification (TSA) using
oligonucleotides labeled with non-radioactive fluorophores is a promising technique for detection and differentiation of fungal
species in environmental or clinical samples, being suitable for microorganisms which are difficult or even impossible to culture.
OBJECTIVE In this study, we aimed to standardise an
in situ hybridisation technique for the differentiation between the pathogenic
species
Paracoccidioides brasiliensis and
Paracoccidioides lutzii, by using species-specific DNA probes targeting the
internal
transcribed spacer-1 (ITS-1) of the rRNA gene.
METHODS Yeast and mycelial phase of each
Paracoccidioides species, were tested by two different detection/differentiation
techniques: TSA-FISH for
P. brasiliensis with HRP (Horseradish Peroxidase) linked to the probe 5’ end; and FISH for
P. lutzii
with the fluorophore TEXAS RED-X
® also linked to the probe 5’ end. After testing different protocols, the optimised procedure
for both techniques was accomplished without cross-positivity with other pathogenic fungi.
FINDINGS The
in silico and in vitro tests show no reaction with controls, like
Candida
and
Cryptococcus
(
in silico) and
Histoplasma capsulatum
and
Aspergillus
spp. (in vitro). For both phases (mycelial and yeast) the
in situ hybridisation showed
dots of hybridisation, with no cross-reaction between them, with a lower signal for Texas Red probe than HRP-TSA probe. The
dots of hybridisation was confirmed with genetic material marked with 4’,6-diamidino-2-phenylindole (DAPI), visualised in a
different filter (WU) on fluorescent microscopic.
MAIN CONCLUSION Our results indicated that TSA-FISH and/or FISH are suitable for
in situ detection and differentiation of
Paracoccidioides species. This approach has the potential for future application in clinical samples for the improvement of
paracoccidioidomycosis patients prognosis.
