Usefulness of molecular biology performed with formaldehyde-fixed paraffin embedded tissue for the diagnosis of combined pulmonary invasive mucormycosis and aspergillosis in an immunocompromised patient

Numerous specimens (more than thirty) were taken from the surgical specimen, fixed in 10% formaldehyde and paraffin embedded. 5 μm paraffin sections were stained with haematoxylin eosin safran (HES), periodic acid Schiff (PAS), Gomori Grocott's methanamine silver (GGM), Gram, Warthin-Starry, and Zielh-Neelsen stains.

Tissue sections were deparaffinized with xylene and DNA was extracted from formalin fixed, paraffin embedded tissue using QIAmp DNA Mini Kit (QIAGEN AG, Hombrechtikon, Switzerland) as described by the manufacturer. The presence of adequate DNA was confirmed by amplifying the human beta-globin gene as previously described [19]. Previously described specific sequences of 5.8 sRNA and 18 sRNA of Aspergillus sp. and mucor, respectively, were chosen as target sequences with slight modification [20, 21]. For the detection of mucor, nested PCR was used with primers described previously [20], but with modification: Mucor1 (5'-WTTACC RTG AGC AAA TCA GA-3') and Mucor2 (5'-CAA TCY AAG AAT TTC ACC TCTAG-3) for the first round and Mucor3 (5'-AGC ATG GAA TAA TRA AAY A-3') and Mucor4 (5'-AGC ATG GGA TAA CGG AAT A-3) for the second round, respectively, giving a final PCR product of 124 bp. For the detection of Aspergillus, the following primers were used as described [21], but with modification: AspNest1 (5'-TCTTGGTTCCGGCATCGAT-3) and AspNest2 (5'TGACAAAGCCCCATACGCT-3') for the first round and AspNest3 (5'-GAAGAACGCAGCGAAATGC-3') and AspNest4 (5'-AACACACAAGCCGTGCTTGA-3') for the second round, respectively, leading to a final PCR product of 146 bp. The basic amplification reactions were done as described before using a volume of 50 μl containing 100 to 500 ng of DNA, 2.5 ml of each primer (final concentration 3 mM), 25 μl Master Mix Gold (Applied Biosystems, Paris, France) [22]. Amplification and detection were performed on the ABI 9800 Fast Thermal Cycler (Applied Biosystem) using for both primer sets, the same cycling conditions with an initial cycling step at 94°C for 10 minutes followed by 94°C, 55°C and 72°C for 30 seconds each ending at 4°C. Ten microliters of each amplification product was analyzed by electrophoresis in an ethidium bromide-containing 2% agarose gel. In addition, PCR products were extracted from a low melting agarose gel and DNA sequencing was performed to confirm the appropriate DNA sequence. Stringent laboratory conditions and appropriate negative and positive controls were performed in each run.

The microscopic examination of the BAL was done in a mixture of potassium hydroxide and chlorazole black E and demonstrated septate and dichotomous branching hyphae. A. fumigatus grew on Sabouraud's glucose agar at 28°C. The strain was tested against voriconazole with the E-test method and showed a good sensitivity [Minimum Inhibit Concentration = 0.125 μg/ml]. The direct examination of the per operative pulmonary biopsy showed non-septate right-angled -branching hyphae suggestive of Mucorales.

All tissue sections stained with HES showed large areas of infarct associated with a few neutrophils, histiocytes and lymphocytes. Venules, arteries and capillaries were congestive or thrombotic. Most of these blood vessels were invaded by large hyphae, measuring from 30 to 50 μm in their largest diameter (Fig. 2A). GGM staining confirmed the presence of numerous fungal elements indicative of a mucormycosis infection: presence of broad-based non septated hyphae with branching at right angles, or twisted hyphae (Fig. 2B). The hyphae were faintly stained with PAS. In a few areas, two different subtypes of hyphae were found in association: first some hyphae were identical to those described above, and second some thinner septated hyphae, 8 to 12 μm in diameter, regular, sometimes branching at acute angles, which could correspond to aspergillosis (Fig. 2C). These filaments invaded blood vessels. Numerous bacteria were observed on HES and Gram staining (Fig. 2C). Other stains gave negative results.

PCR analysis from deparaffinized sections performed from a selected paraffin block showing both subtype of hyphae, allowed identification of aspergillosis and mucormycosis (Fig. 2D). DNA sequencing of the PCR products were performed and showed a 100% concordance with Aspergillus sp. and Rhizopus sp. (namely Rhizopus microsporus) for the apergillus and mucor PCR, respectively.

Culture on Sabouraud's dextrose agar at 28°C yielded a fast-growing mold (3 days). Colonies were woolly and initially white, quickly become grayish brown. The fungus was identified as Rhizopus microsporus var. rhizopodiformis on the basis of morphological features: rhizoids and sporangiophores of 400 μm in length and in groups of 1 to 4; sporangia spherical of approximately 100 μm grayish black; spherical sporangiospores (5 μm) fine spinulose and the capacity to grow at 50°C (Fig. 3A-C). The morphological identification was confirmed by sequencing the whole ITS1-5.8S-ITS2 region [23]. Antifungal sensitivity testing using the EUCAST method showed a decreased susceptibility to the voriconazole (MIC = 8 μg/ml); and a MIC that was considered to be within the sensitivity range to amphotericin B (MIC = 0.06 μg/ml) and posaconazole (MIC = 0.5 μg/ml).