Molecular Investigation for Bacteria
Due to the finding of granulomas revealed by the histopathological investigation, the presence of mycobacterial DNA was investigated in frozen (pool made of liver, spleen, heart, and kidney), and in formaldehyde‐fixed paraffin‐embedded (FFPE) samples. Frozen samples were processed for DNA extraction using the Purelink Genomic DNA kit (Invitrogen, USA) following the manufacturer’s instructions. FFPE samples were processed for DNA extraction using the Purelink Genomic DNA kit (Invitrogen, USA) following the manufacturer’s instructions with minor modifications. Particularly, unstained sections, serial to sections showing granulomas, were used for DNA extraction: 5–10 mg of sliced FFPE tissue was placed in 1 mL of xylene (J.T. Baker) and a pre-extraction step to remove paraffin from the sample was applied as previously described (Sirri et al., 2018). Samples were deparaffinized in xylene for 5 min; following centrifugation, samples were washed twice in 100% Ethanol. The pellet was dried at 37 °C for 10 minutes, and DNA extraction was subsequently undertaken using the aforementioned kit. Bacterial presence was investigated through a PCR method targeting the HSP65 gene using primers common to all mycobacteria (Telenti et al., 1993). For the amplification, a reaction mixture was assembled containing 1X PCR Buffer, 1 mM MgCl2, 200µM dNTP, 0.4 µM of each primer, 2.5U of Taq polymerase and 5µl of DNA in a final volume of 25 µl. The amplification cycle was conducted at 94 °C for one minute, 45 cycles consisting of denaturation at 94 ° C for one minute, annealing at 60 °C for one minute, extension at 72 ° C for one minute and a final extension step at 72 °C for 10 minutes. The PCR results were visualized by electrophoresis on 1.5% agarose gel by running the samples with a reference marker (100 bp ladder Invitrogen). PCR products of positive samples were purified using the ExoSAP-IT PCR Product Cleanup Reagent (Invitrogen, USA), and sequenced through the Bio‐Fab Sequencing Service (Rome, Italy). The sequences were then manually corrected and subjected to BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for identification.