First Human Case of Pulmonary Fungal Ball Due to a Perenniporia Species (a Basidiomycete)

Mycological investigations.Direct microscopy of KOH wet mounts of the BAL fluid specimen revealed hyaline septate hyphae (Fig. 2A). Cultures yielded multiple white, cottony colonies of identical molds on Sabouraud's glucose agar (SGA) plates incubated for 7 days at 28°C and 37°C. Subcultures on potato dextrose agar (PDA) incubated at 28°C and 37°C showed dense white cottony growth after 7 days (Fig. 2B), and slide cultures of the mold isolates on PDA at 28°C revealed hyaline septate hyphae with chlamydospore-like cells (Fig. 2C). No clamp connections or hyphal pegs were seen during up to 3 weeks of incubation. The isolate was assigned accession no. VPCI 85/P/10 (CBS 130020) for molecular identification and antifungal susceptibility testing. BAL fluid was also investigated microscopically after Gram and Ziehl-Neelsen (ZN) staining and cultured for aerobic pathogens and Mycobacterium spp. The Gram and ZN stains were negative, and cytology was negative for any malignant cells. No Mycobacterium spp. were isolated after 6 weeks of incubation.

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Fig 2

(A) KOH wet mount of BAL fluid specimen of the patient showing hyaline septate hyphae (magnification, ×400). (B) White cottony growth on PDA culture plate seen after 7 days of incubation at 28°C. (C) Slide culture of the isolate on PDA showing hyaline septate hyphae with chlamydospores (magnification, ×400). (D) Ouchterlony's agar gel double diffusion test of the patient's serum showing two precipitin bands against a Perenniporia species isolate (a) and negative results with antigens of A. fumigatus (b), A. flavus (c), and A. terreus (d).

Immunodiffusion of the patient's serum demonstrated precipitins against culture filtrate antigen prepared from the patient's isolate as described previously (Fig. 2D) (3, 4). In contrast, negative results were found in tests using antigens of Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, and Aspergillus niger. It was evident from these findings that the patient was suffering from posttuberculosis fibrocavitary disease of the right lobe with a fungal ball. Since no episodes of hemoptysis in the recent past were reported by the patient, no active intervention was done and he was managed conservatively on an outpatient basis. He was briefed about the possibility of recurrent episodes of hemoptysis, for which he was prescribed ethamsylate and cough suppressants, and was advised to consult a nearby medical facility for the same in his hometown in Assam.

The identification of 85/P/10 (CBS 130020) was done by sequencing of the internal transcribed spacer (ITS) ribosomal DNA (rDNA) region and D1/D2 large subunit (LSU) regions as described previously (3, 4). GenBank BLAST searches were performed for species identification. The LSU sequence of CBS 130020 showed 99% identity with Megasporoporia setulosa GU566007 (strain MG38) and 100% identity with an unidentified Perenniporia species, strain 1V2/2 (GQ982883). The ITS region sequence of the isolate exhibited 100% identity with the Perenniporia strain 1V2/2 (GQ982890). The nearest neighbor, Megasporoporia setulosa JF894111, showed 90% similarity. The ITS and LSU nucleotide sequences for the isolate CBS 130020 were deposited in GenBank under the accession numbers JX271779 and JX292098, respectively.

ITS sequences from reference isolates belonging to the genera Abundisporus (n = 5), Agaricomycetes (n = 2), Donkioporia (n = 1), Megasporoporia (n = 6), Microporellus (n = 1), Perenniporia (n = 53), Perenniporiella (n = 7), Polyporus (n = 1), Pyrofomes (n = 1), and Trametes (n = 1), described by Guglielmo et al. (14), Robledo et al. (23), Pinruan et al. (20), Yuan et al. (32), and Zhao and Cui (33), were included in the analyses (Table 1 ). Evolutionary analyses were conducted in MEGA5 (30) with the maximum likelihood method. Evolutionary distances were computed using the Kimura 2-parameter method (16) with 1,000 bootstrap replicates (10). A discrete gamma distribution was used to model evolutionary rate differences among sites.

The complete alignment included 79 sequences. Aligned sequences of the ITS were 866 bp long, including 340 invariable characters, 324 variable parsimony-informative (37.4%) characters, and 108 singletons. Positions containing gaps and missing data were eliminated. The tree comprised 31 described Perenniporia species, including the generic type species P. medulla-panis. All of the unresolved deeper branches exhibited bootstrap values below 80%, raising concern about the cluster of related species. Furthermore, the tree (outside the ancestral Abundisporus branches) included species of Donkioporia, Megasporoporia, Polyporus, Pyrofomes, and Trametes, which may indicate possible misidentification. The clinical isolate CBS 130020 shared a supported clade (bootstrap, 94%) with unidentified Agaricomycetes and a Megasporoporia sp. and was found to be identical to the unnamed basidiomycetous endophyte 1V2/2, described by Pinruan et al. (20) (Fig. 3). The nearest taxa are Perenniporia subacida and Perenniporia narymica, located in a sister clade at a 90% bootstrap level.

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Fig 3

Molecular phylogenetic tree based on ITS sequences generated in this study by the maximum likelihood method based on the Kimura 2-parameter model. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches (bootstrap support values of >90% are indicated in bold). GenBank accession numbers are indicated before the strain code.

Antifungal susceptibility testing (AFST) of the isolate was performed by the CLSI broth microdilution method (5). The antifungals tested were amphotericin B (Sigma, St. Louis, MO), fluconazole (Pfizer, Groton, CT), itraconazole (Lee Pharma, Hyderabad, India), voriconazole (Pfizer), posaconazole (Schering-Plough, Kenilworth, NJ [now Astellas]), isavuconazole (Basilea Pharmaceutica, Basel, Switzerland), flucytosine (Sigma), caspofungin (Merck, Whitehouse Station, NJ), micafungin (Astellas, Toyama, Japan), and anidulafungin (Pfizer). For the broth microdilution test, RPMI 1640 medium with glutamine without bicarbonate (Sigma) buffered to pH 7 with 0.165 mol/liter 3-N-morpholinepropanesulfonic acid (Sigma) was used. Isolates were grown on PDA for 8 days at 37°C, and the inoculum was adjusted to a final density of 1.0 × 10⁴ to 5.0 × 10⁴ hyphal fragments/ml measured by a spectrophotometer. Drug-free and mold-free controls were included, and microtiter plates were incubated at 35°C for 72 h. CLSI-recommended quality control strains Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 and reference strains Aspergillus fumigatus ATCC 204305 and Aspergillus flavus ATCC 204304 were included. The MIC endpoints were read visually; for azoles and amphotericin B, MICs were defined as the lowest concentration at which there was 100% inhibition of growth compared with the drug-free control wells. For echinocandins, minimal effective concentrations (MECs) were defined as the lowest concentration of drug that led to the growth of small, rounded, and compact hyphal forms. For the isolate, the lowest MIC was that of posaconazole (0.06 μg/ml), followed by itraconazole (0.5 μg/ml), voriconazole (2 μg/ml), and isavuconazole (2 μg/ml). Amphotericin B had a MIC of 0.25 μg/ml. All three echinocandins showed good activity (MECs, 0.125 to 0.5 μg/ml). Fluconazole and flucytosine did not show any activity (64 μg/ml).

The clinical significance of white, cottony, rapidly growing filamentous molds from pulmonary samples is poorly understood. In the past, cultures have often been discarded as purported contaminants, because morphological identification of these nonsporulating fungi was impossible, and hence, they could not be attributed to any of the known pathogens. Today, we know that many of these cultures are of basidiomycete affinity (9). The most common of these is Schizophyllum commune, which is recognizable morphologically by the presence of hyphal pegs (26) and sometimes by clamp connections and by the formation of abortive fruiting bodies (3). Occasionally, another basidiomycete repeatedly isolated from pulmonary infections in humans is Hormographiella aspergillata, the anamorph of the Agaricales mushroom Coprinopsis cinereus (11, 13, 27, 28, 31). The Coprinopsis species are recognizable in culture by the formation of arthroconidial anamorphs (9). Recently, some lesser-known basidiomycete species, such as Cyclomyces tabacinus (18), Irpex lacteus (2), Inonotus (Phellinus) tropicalis (7, 29), Oxyporus corticola (1), and Volvariella volvacea (25), have been added as causative agents of pulmonary and fatal deep-seated mycoses in humans and animals. Other basidiomycetes such as Phanerochaete chrysosporium (anamorph, Sporotrichum pruinosum) and Bjerkandera adusta have repeatedly been isolated from pulmonary sites and may also be pathogenic in some settings (12, 15).

The majority of infections caused by filamentous basidiomycetes are associated with chronic colonization of cavities in lungs or sinuses (3, 21, 26). Occasionally, however, this may lead to fatal dissemination and cerebral involvement, implying that this fungal group may have a neurotropic potential (22). As long as there is a paucity of information on fungus and host responses for filamentous basidiomycetes, infections by these fungi should be treated with caution. The present case demonstrates a novel agent of fungal ball due to a Perenniporia species. The case was diagnosed by CECT showing intracavitary mass, by bronchoscopy, by direct demonstration (KOH wet mount), and by isolation of the species in culture from BAL fluid. Serological analysis demonstrated precipitins against the etiologic agent. Many patients with fungal ball are asymptomatic, but the most frequent symptom is hemoptysis. Less commonly, patients develop chest pain, dyspnea, malaise, and wheezing. In the present case, the patient was afebrile with a history of mild hemoptysis. In asymptomatic patients, no treatment is required, regular observation being sufficient in most cases. There is no consistent evidence that fungal ball responds to antifungal agents, and such drugs rarely achieve the effective concentrations within the lung cavities (19).

The isolate did not show the characteristics that facilitated recognition of a filamentous basidiomycete such as the presence of clamp connections and/or crystals, the formation of spicules along the hyphae, and mushroom- or basidiocarp-type fruiting bodies. Since most clinical isolates are monokaryons, neither clamps nor fruiting structures are produced. The isolate was proven to be affiliated with the filamentous basidiomycetes through sequencing. Definite identification was impossible, because it showed 100% identity with an as-yet-unidentified species attributed to Perenniporia and it was close to several unnamed basidiomycete species (Fig. 3). The nearest named species was Megasporoporia setulosa, at a 10% ITS distance. Taxonomically, Perenniporia and Megasporoporia belong to the order Poriales in the Agaricomycetes, Basidiomycota. Fungi of this group are characterized morphologically by formation of porate, often resupinate fruiting bodies which are flat on the substrate with the hymenium on the outer side on rotten branches. The genus Perenniporia contains numerous species growing as saprobes on dead wood (6, 33). They are ubiquitously present under almost all types of climatic conditions, degrading wood by decomposition of lignin and, to a limited extent, cellulose, leading to white rot. Most species are presently known from herbarium materials only and have not been sequenced. The fact that the sequence of our strain did not have a match in GenBank is therefore not surprising, as there are not enough data in GenBank to identify unknown sterile basidiomycetes with a high degree of confidence by ITS and/or D1/D2 sequencing (24). Through GenBank, similarities also were found with other genera of shelf fungi, such as Trametes, Donkioporia, and Pyrofomes (Fig. 3). The taxonomy of these fungi has insufficiently been resolved using modern, nonmorphological techniques, and the sequences available thus far do not clearly resolve the genera (Fig. 3). Therefore, description of our strain as a novel species in any of the genera mentioned does not seem to be appropriate. In our tree (Fig. 3), the name Perenniporia is widely distributed (31 described species). The generic type species, P. medulla-panis, included in the tree is represented by strains studied by Decock and Stalpers, who rectified the generic typification (8). This and several other Perenniporia species show variability in the sequenced gene. Perenniporia medulla-panis occupies a central location compared to the remaining species, and the main branches lack statistical support, indicating concerns due to related taxa. Therefore, attribution of our strain to the genus Perenniporia is justified.

Perenniporia species have never been reported as etiologic agents of human disease before. Similarly to Schizophyllum, the fungus produces large amounts of airborne basidiospores which are easily inhaled. The frequency with which this leads to colonization of pulmonary cavities or systemic dissemination is presently unknown. This report extends the genera of basidiomycetous fungi implicated in pulmonary infections and underscores the utility of molecular methods in the identification or verification of these often unidentifiable molds when conventional mycological techniques fail to identify the incriminated fungi (21).

Nucleotide sequence accession numbers.The ITS and LSU nucleotide sequences for the isolate CBS 130020 were deposited in GenBank under the accession numbers JX271779 and JX292098, respectively.