Evidence Implicating Thamnostylum lucknowense as an Etiological Agent of Rhino-Orbital Mucormycosis

Abstract

In this report, we present a case of rhino-orbital mucormycosis in a 57-year-old female with poorly controlled diabetes mellitus. The only mold cultured at 25°C, 37°C, and 40°C from a specimen of the nasal crust was identified phenotypically and independently using nuclear ribosomal DNA sequence data as Thamnostylum lucknowense. To our knowledge, this report presents the first data implicating this mucoraceous fungus as a mycotic agent of human infection.

CASE REPORT

A 57-year-old female patient was admitted to the Emergency Department of the All India Institute of Medical Sciences, New Delhi, India, in February 2009 with complaints of swelling and loss of vision in the right eye over the previous 10 days and right-sided nasal obstruction for the previous week. She had a history of fever, vomiting, and itching in the right eye, with intermittent episodes of altered sensorium. Her current symptoms started with itching followed by loss of vision in the right eye and nasal obstruction and then altered sensorium. She had poorly controlled diabetes mellitus for the previous 8 years and hypertension for 5 years. Her blood pressure was controlled with oral amlodipine at 5 mg daily. There was no history of seizures or trauma.

On examination, the patient was disoriented. Her heart rate was 120/min, and blood pressure was 160/70 mm Hg. Ophthalmological examination revealed hyperemia, proptosis, discharge, and sluggish papillary reaction to light in her right eye. Her random blood sugar was 19.7 mmol/liter. She was treated initially with ceftazidime (1 g) every 12 h, vancomycin (500 mg) infusion every 6 h, and subcutaneous injection of human insulin (Actrapid). The patient was transferred to the otorhinolaryngology department for further management.

A contrast-enhanced computed tomographic (CT) scan of her brain and orbit showed bilateral polypoidal opacification of the maxillary, sphenoid, and ethmoid sinuses together with involvement of the cavernous sinus. A specimen of the nasal crust was sent to the microbiology laboratory for testing. Direct examination of a portion of the nasal crust in a KOH mount using light microscopy showed hyaline, coenocytic hyphae 4.0 to 7.0 μm in diameter. A portion of the nasal crust was cultured on Sabouraud dextrose agar with and without chloramphenicol at 25°C, 37°C, and 40°C in the dark. After 48 h of incubation, only white, cottony colonies producing aerial hyphae were observed on slant cultures at each temperature. No bacterial growth was evident. Over the next 2 to 3 days, the colonies turned from white to olive brown and filled the culture tubes. The in vitro MICs of the following four antifungals were determined by the broth microdilution technique according to the CLSI M38-A2 protocol (7): amphotericin B, 0.5 μg/ml; itraconazole, 0.5 μg/ml; posaconazole, 0.25 μg/ml; and voriconazole, >64.0 μg/ml.

Once the diagnosis of acute rhino-orbital mucormycosis was made, the patient was treated with an intravenous infusion of conventional amphotericin B deoxycholate at 50 mg/day. However, the patient developed features of renal failure within a week, with urea and creatinine levels increasing to 28.6 mmo/liter and 194.5 μmol/liter, respectively. The dose of amphotericin B was reduced to 25 mg/day on the sixth and seventh days. However, she developed hemodynamic instabilities and became unconscious. Subsequently, due to respiratory distress, a tracheostomy was performed and she was put on mechanical ventilation. Although surgical debridement and biopsy for histopathological study was planned, it could not be undertaken in view of the deteriorated condition of the patient. On the 10th day of hospitalization, the patient died due to cardiac arrest and multiorgan failure.

Fungal identification.

A subculture was sent to Gerald L. Benny, Department of Plant Pathology, University of Florida, Gainesville, for morphological analysis and to the National Center for Agricultural Utilization Research (NCAUR), ARS-USDA, Peoria, IL, where it was accessioned in the ARS Culture Collection (http://nrrl.ncaur.usda.gov/) as NRRL 54533 for molecular phylogenetic analysis. For the phenotypic studies, the mucoraceous mold was cultured on malt extract agar (MEA; Difco, Detroit, MI) and potato dextrose agar (PDA; Difco, Detroit, MI) at 25°C in the dark. Colonies were fast-growing, initially white and cottony, reaching a diameter of approximately 10 cm after 7 days incubation. Colonies gradually turned pale yellow to olive brown after 7 days at 25°C on MEA and PDA. Aerial sporangiophores were produced from the substrate mycelium and stolons; these usually produced a terminal sporangium and a subterminal cluster of obpyriform sporangiola. Dehisced sporangia mounted in 2% KOH–phloxine in distilled water (3) bore a distinct columella and a collar (Fig. 1A). Whorls of obpyriform, recurved pedicillate sporangiola were typically formed on subterminal vesicles on the sporangiophores (Fig. 1B to D). Sporangiola mostly produced 8 to 16 sporangiospores that were oval, smooth, and hyaline and measured 3 to 7 by 2 to 3 μm. Based on this analysis, NRRL 54533 was identified as Thamnostylum lucknowense (J. N. Rai, J. P. Tewari & Mukerji) Arx & H. P. Upadhyay (4), a species that was originally isolated from soil in Lucknow, India, and designated Helicostylum lucknowense (16). Subsequently, Von Arx and Upadhyay transferred this species to Thamnostylum (20) based on the shared production of stolons and rhizoids, apophysate sporangia, and pyriforme sporangiola borne on circinate stalks.

Fig 1.

Microscopic features of Thamnostylum lucknowense NRRL 54533. (A) Columella of terminal sporangium, showing collar (arrow). Bar, 20 μm. (B) Fertile head of immature sporangiola on fertile vesicle, showing recurved and unbranched pedicels. Bar, 20 μm. (C) Fertile head of mature sporangiola. Bar, 20 μm. (D) Typical fertile vesicle bearing pedicellate sporangiola. Bar, 50 μm.

Methods to obtain multilocus DNA sequence data, including extraction of total genomic DNA, PCR amplification, and DNA sequencing, followed published protocols (11, 14, 15). Briefly, isolate NRRL 54533, and the ex-type strains of Thamnostylum nigricans NRRL 6243, T. piriforme NRRL 2342, and T. lucknowense NRRL 2892 and the ex-neotype strain of T. repens NRRL 6241, were cultured in yeast-malt broth (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 2% dextrose; Difco, Detroit, MI) and then harvested over a Büchner funnel. The mycelium was freeze-dried overnight, and then genomic DNA was extracted using a CTAB (hexacetyltrimethylammonium bromide; Sigma Chemical Co., St. Louis, MO) Miniprep apparatus (14). Published PCR primers were used to amplify and sequence the nuclear small subunit (SSU 18S) ribosomal DNA (rDNA) (21) and domains D1 and D2 of the nuclear ribosomal large subunit (LSU 28S) ribosomal DNA (15). ABI chromatograms were edited with Sequencher version 4.1.2 (Gene Codes, Ann Arbor, MI), exported as NEXUS files, and then aligned with published 18S and 28S rDNA data sets for the Mucorales (15) by the use of MUSCLE (10).

Preliminary maximum parsimony (MP) analyses (18) of the individual and combined two-locus data sets indicated that NRRL 54533, and the other Thamnostylum species sequenced, were nested within clade 2 of the Mucorales as defined in O'Donnell et al. (15). Therefore, a subset of sequences comprising nine clade 2 taxa was constructed and analyzed using MP and maximum likelihood (22). The results of these analyses strongly indicated that NRRL 54533 and the ex-type strain of T. lucknowense NRRL 2892 were conspecific (Fig. 2).

Fig 2.

Maximum parsimony (MP) bootstrap consensus phylogram showing case report isolate NRRL 54533 grouping with the ex-type strain (T) of Thamnostylum lucknowense NRRL 2892 with strong MP (above node) and maximum likelihood (ML; below node) bootstrap support (MP = 99%; ML = 98%). MP and ML bootstrapping involved 1,000 pseudoreplicates of the data. The phylogram represents the single-most-parsimonious tree inferred from the combined nuclear ribosomal 18S rDNA (1,719-bp alignment; 96 parsimony-informative characters [PIC]) and partial nuclear ribosomal 28S rDNA (616-bp alignment; 85 PIC). Note that Thamnostylum appears to be paraphyletic given that the ex-type strains of Fennellomyces linderi and Zychaea mexicana are nested within it. Sequences of Phascoloymces articulosus NRRL 2880 and Circinella umbellata NRRL 1351 were used to root the phylogram by the outgroup method based on a more inclusive analysis (15). CI, consistency index; T, ex-type strain; NT, ex-neotype strain.

Discussion.

Herein we report the first evidence to implicate Thamnostylum lucknowense as a human mycotic agent. This inference is based on the fact that hyaline, coenocytic hyphae were detected by direct microscopy in the nasal crust specimen; this fungus was the only mold cultured from the patient's nasal cavity and grew well at 37°C and 40°C. Although rhino-orbitocerebral infections caused by mucoraceous molds are the most common mycoses among Indian patients (5, 6, 9), only about one-fifth are cultured and identified at the genus level (11), suggesting that novel etiologic agents are likely overlooked in India in the absence of detailed morphological and/or molecular phylogenetic data confirming the identification. Note that our data strongly suggest but do not confirm that T. lucknowense caused the infection, because no cultures were made using tissue samples from deep within the patient and an autopsy was not performed. As is common with rhino-orbital infections (9, 12, 13, 17), our patient had the predisposing condition of uncontrolled diabetes mellitus. She presented with symptoms consistent with rhino-orbital mucormycosis, including fever, nasal obstruction, orbital proptosis with swelling and painful erythema, and loss of vision. CT radiography revealed intraorbital inflammation and sinus opacification, classic hallmarks of a rhino-orbital infection (2, 12, 13). Identification of broad coenocytic hyphae in a specimen of the nasal crust, from which the mucoraceous mold was subsequently cultured, facilitated its identification as a mucoraceous fungus (8). As practiced routinely in India to treat invasive mucormycosis in a resource-limiting situation, the patient was treated with the recommended dosage of amphotericin B deoxycholate for the first 5 days. Subsequently, a MIC of 0.5 μg/ml of amphotericin B deoxycholate against the isolate supported the choice of this antifungal agent (13). However, as often reported (1, 17), the patient poorly tolerated this therapy and experienced nephrotoxicity and renal failure. The patient succumbed to her illness before modification of the antifungal therapy. For future reference, results of our in vitro susceptibility testing indicated that two different azoles with low MICs, posaconazole (0.25 μg/ml) and itraconazole (0.5 μg/ml), might have been efficacious in vivo against our isolate of T. lucknowense (1).

Our working hypothesis that is our patient inhaled spores from soil or rodent dung, the only known environmental reservoirs of T. lucknowense (16, 19). This species was initially isolated from soil in Lucknow, India, and described as a new species within Helicostylum (16). Subsequently, it was isolated in California from pack rat and mouse dung (19). Based on the results of a comparative morphological study, H. lucknowense, H. piriforme, and H. repens were later transferred to Thamnostylum (20). The molecular phylogenetic results strongly support the morphological identification as T. lucknowense. In addition, this analysis also indicated that Thamnostylum is paraphyletic, given that two monotypic genera, Fennellomyces linderi and Zychaea mexicana, are nested within it (4). Synapomorphies that unite these taxa include the production of rhizoids, stolons, and pedicellate, collumellate, apophysate sporangiola with few spores. Zychaea mexicana and F. linderi appear to represent Thamnostylum species that have lost, respectively, the ability to produce multispored sporangia and vesicles bearing pedicellate sporangiola. The paraphyly of Thamnostylum is consistent with a more inclusive multilocus molecular phylogenetic study of the Mucorales that discovered that many families and several of the larger genera were poly- or paraphyletic (15).

Nucleotide sequence accession numbers.

The nucleotide sequences generated in this study were deposited in GenBank under accession numbers JQ043221 to JQ043226.