This was a cross-sectional study of a contemporary cohort.

Palatine roots of first maxillary molars that were radiographically in close contact with maxillary sinus, with endodontic treatment performed over a 3-year period prior to the study and with evidence of apical periodontitis.

Teeth with pulp chamber exposed to oral cavity, with symptoms of pain, presence of mobility, and periodontal pocket. Patients treated with oral antibiotics and/or antifungals in the last six months and those with clinical manifestations of oral candidiasis were also excluded.

The selected teeth were accessed after receiving absolute isolation, antisepsis with 5.25% sodium hypochlorite, and neutralization with 5% sodium thiosulfate. Access to root canals was performed with the use of a high-speed, carbide, round drill bit, and then the operative field was again cleaned with the aforementioned solutions. The palatine root canal was irrigated with sterile saline; according to a pre-established length calculated in an initial radiography, gutta-percha was removed without using solvent, using Gates-Glidden burs #2 and #3 and Hedströen files #25, #30 and #40. Then, a Kerr file of compatible diameter and length for the root canal was passively introduced, and the odontometry was established with the use of an apex locator, with radiographic confirmation.

Following the protocol recommended by Gomes et al.,10 the channel was irrigated with sterile saline and a Hedströen file #40 was used to remove dentin shavings. Then, three sterile absorbent paper points were inserted into the root canal, one by one, 1mm short of the root apex, and maintained there for one minute. These paper points were then transferred to a test tube containing Sabouraud agar (Difco Laboratories, Detroit, MI, United States) with addition of 0.5% chloramphenicol (Medleyk Campinas, SP, Brazil), in a two-lamp isolated field. An open Petri dish containing the same culture medium (Sabouraud agar plus 0.5% chloramphenicol) was placed within the field isolated by the two lamps in order to verify the accuracy of isolation of the operative field (negative control). At the same time, a positive control, including a Petri dish containing the same culture medium but which remained open outside the isolated field during the collection of samples, was conducted, in order to check the growth of potential environmental fungi, verifying whether these organisms coincided with those isolated from root canals. Petri dishes and test tubes were kept at room temperature for 7–14 days; during this period, mycelial growth was observed for subsequent isolate analysis at the Fungal Culture Collection Laboratory.

From a monosporic culture, a colony fragment was collected and placed on a slide. A drop of Amann's lactophenol-cotton blue cytoplasmic stain was added (Difco Laboratories, Detroit, MI, United States) and an optical microscope (YS 100; Nikon Corporation, Japan) was used to observe vegetative and reproductive mycelium of the fungi.

After checking the characteristics of the fungi, the strains were subcultured with the aid of a sterile platinum loop, and one fragment of fungus was inoculated on a Petri dish containing 20mL of a specific medium to facilitate its growth and sporulation. Depending on the characteristics presented, a specific culture medium was used, including malt extract agar, Czapek agar, or potato agar (Difco Laboratories, Detroit, MI, United States). The colonies were incubated at room temperature and observed every 24h, being aware that each strain has a specific growth time, depending on its taxonomic group. Then, the colonies were analyzed and classified macroscopically with respect to texture, color (front and back side), diameter, pigment production, growth time, and production or nonproduction of exudate.

Mycelial fragments of each isolate were carefully examined in preparations obtained from microcultures in a specific medium, according to the Rivalier and Seydel technique.17 This methodology shows the arrangement of conidia in situ, allowing the morphological visualization of reproductive and/or propagation structures under optical microscopy (YS 100; Nikon Corporation, Japan), after slide preparation and application of a cytoplasmic stain, Amann's lactophenol-cotton blue (Difco Laboratories, Detroit, MI, United States). From morphological macroscopic and microscopic studies and with the aim of the specific literature, the taxonomic identification of isolated fungi was performed.18–20

Aiming to confirm the taxonomic identification, sequencing of isolates was performed. DNA extraction was performed according Yamakami et al.21 Approximately 50mL of culture medium were inoculated with a conidium suspension and incubated at 30°C×72h. The mycelium was transferred to a 50-mL polypropylene vial containing six glass spheres (diameter 4mm). The tube was immersed in liquid nitrogen for 10s and agitated in a vortex for 20s. Then, 2mL of DNA extraction buffer (containing 10mM Tris–HCl [pH 8.0], 10mM EDTA, 0.15M NaCl, 2% sodium dodecyl sulfate [SDS], and 0.5mg proteinase K per mL [Sigma Chemical Co., St. Louis, MO, United States]) were added. Then, the mixture was allowed to stand for thawing. This mixture was incubated at 55°C×2h. Proteinase K was inactivated by heating the mixture at 95°C×10min, and a volume of 0.7mL was transferred to a 1.5-mL microcentrifuge tube; and an equal volume of phenol–chloroform (1:1) was added. The mixture was centrifuged at 10,000g at 4°C×5min. The supernatant was transferred to another tube and the same procedure was repeated with chloroform–isoamyl alcohol (24:1). The DNA was precipitated with two volumes of ethanol at −20°C and centrifuged at 12,000×g at 4°C×20min. Thus, the pellet obtained was dried. After washing with 70% ethanol at 4°C, the extracted DNA was dissolved into 50μL of distilled water and 5μL of suspension, and used for polymerase chain reaction (PCR).

Amplification reactions were performed in a final volume of 25μL containing 10pmol of each primer; 1.5mL of MgCl2 (25mM); 1.0μL of dNTPs (5mM); 2.5μL of 10× buffer (Taq); 1 unit of Taq DNA polymerase, and 20ng of DNA (Fermentas Inc.). Primers ITS5 and ITS4 (ITS5 – 5′-GGA AGT AAA AGT CGT AAG AAC G-3′, ITS4 – 5′-TCC TCC GCT TAT TGA TAT GC-3′) were used. PCRs were performed in a T100 thermocycler (Bio-Rad Laboratories Brasil Ltda.), programmed with the following steps: pre-denaturation at 94°C×5min, 30 cycles (denaturation at 94°C×30s), 40–55°C with an increase of 0.5°C/s (totaling 30s) (annealing), and extension (72°C/1min×30s). The final extension was carried out at 72°C×4min (29). The amplification reaction products were detected using agarose gel electrophoresis in 1% 1× TBE buffer (Tris–borate EDTA), and an electrophoresis analysis was performed at 36V×2h. A 1-Kb DNA molecular marker was used. The gel was stained with ethyl bromide 0.5μg/mL for 15min, visualized with the aid of an ultraviolet (UV) transilluminator, and photographed with an image detection and analysis automatic system (Gel Doc XR+System with Software; ImageLab, Bio-Rad Laboratories Brasil Ltda.).

RCP products (6μL) were treated with the enzymes exonuclease I (USB; 3.3L) and shrimp alkaline phosphatase (SAP, USB; 0.66L) in order to eliminate remnants of primers and of dNTPs. The tubes containing the mixture were maintained at 37°C×45min and then at 80°C×15min. In the sequencing reaction, fluorescent dideoxynucleotide was used with an ABI Prism 377 automatic sequencer model. The reactions were performed in a final volume of 10μL containing 0.5mL of primers DYEnamic ET Terminator Sequence Premix (DYEnamic ET Dye Terminator Cycle Sequencing Kit; Amersham Bioscience). The T100 thermocycler (Bio-Rad Laboratories Brasil Ltda.) was programmed in 30 cycles of 96°C×30s, 40°C×30s, and 60°C×1min. An aliquot (20μL) of the sequencing reaction was transferred to a sterile tube, with addition of 20μL of MilliQ® water and 60μL of isopropanol. The mixture was homogenized by vortex, left for 20min at room temperature, and then centrifuged. The supernatant was removed and the precipitate washed twice with 250μL of 70% ethanol and dried in an oven at 37°C. The DNA was dissolved in 4μL of Formanide Loading Dye and subjected to electrophoresis in an ABI PRISM 377 automatic sequencer (Applied Biosystems). The obtained sequences were analyzed using the Basic Local Alignment Search Tool program (BLASTn) and the sequences obtained were compared with those deposited at GenBank.