JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/01/$04.00�0 DOI: 10.1128/JCM.39.11.4213–4218.2001

Nov. 2001, p. 4213–4218 Vol. 39, No. 11

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Isolation of a Nodulisporium Species from a Case ofCerebral Phaeohyphomycosis

P. UMABALA,1 V. LAKSHMI,1* A. R. MURTHY,2 V. S. S. V. PRASAD,2 C. SUNDARAM,3 AND H. BEGUIN4

Departments of Microbiology,1 Neurosurgery,2 and Pathology,3 Nizam’s Institute of Medical Sciences, Hyderabad,Andhra Pradesh, India, and Scientific Institute of Public Health, BCCM/IHEM Culture Collection, Brussels, Belgium4

Received 16 March 2001/Returned for modification 30 May 2001/Accepted 8 July 2001

A fungal infection of the brain of a 55-year-old male patient is reported. The lesion and involved fungus werelocated exclusively in the right medial temporo-parietal region. The patient was successfully treated withsurgical resection of the lesion and antifungal chemotherapy. Few pathogenic dematiaceous fungi exhibitneurotropism and can cause primary infection in the central nervous system (CNS). The etiological agent isdescribed as a Nodulisporium species. To date Nodulisporium has never been reported as an agent of CNSinfection in humans.

CASE REPORT

A 55-year-old male patient was admitted to the neurosur-gery facility of Nizam’s Institute of Medical Sciences (NIMS),Hyderabad, Andhra Pradesh, India, with an admitting diagno-sis of right choroidal meningioma.

Two months prior to admittance to NIMS, the patient ex-perienced rapid deterioration of vision in the right eye alongwith associated numbness in the right half of the face. For 1year prior to admittance he had also experienced difficulty inchewing food and intermittent episodes of transient loss ofconsciousness associated with weakness on the right side,mainly involving the limbs. The recovery time from such epi-sodes was about 1 to 2 h. There was no history of generalizedtonic or clonic seizures. He did not have a history of hyperten-sion or diabetes and was not on any medication.

A computerized tomogram (CT) of the brain was performedby the referring hospital (Fig. 1). CT sections 5 mm thick wereobtained in the posterior fossa, and CT sections 10 mm thickwere obtained thereafter, both before and after administrationof intravenous contrast (ionic contrast [40 ml of 76% UroVideo; Bracco]). The scan revealed a large, irregularly shaped,slightly hyperdense, densely enhancing lesion in the right me-dial temporo-parietal region of about 44.3 by 33.2 mm. Itshowed a large, irregular, hypodense white matter with sur-rounding edema. There was a shift in the midline structures tothe left. The ventricular system and sulcal cerebrospinal fluidspaces were effaced more on the right. The overall impressionwas of a sphenoid meningioma in the right medial temporo-parietal region with cerebral edema. With these clinical details,the patient was referred to NIMS for further management.

The patient was conscious and coherent. There were nosigns of anemia, clubbing, or palpitation. His pulse and respi-ratory rate were within the normal range. There was no lymph-adenopathy or organomegaly.

The right eye showed exophthalmos, with no pupillary light

reaction and primary optic atrophy on fundus examination.The left eye was normal. Paresis of the right fifth and sixthcranial nerves was present. There was loss of motor and sen-sory components of the fifth cranial nerve. There were noother sensory or motor deficits.

The peripheral blood picture and biochemistry were withinnormal limits. Hemoglobin was 13.5 g%, packed cell volumewas 39%, and total leukocyte counts and differential countswere within normal range. The test of anti-human immunode-ficiency virus antibodies was nonreactive. Blood urea and se-rum creatinine were 45 and 0.7 mg%, respectively. The chestradiograph and two-dimensional echocardiogram of the heartwere normal. With a diagnosis of right choroidal meningioma,the patient was scheduled for surgery and excision of the tu-mor.

The tumor was approached by a right temporal craniotomy.During the operation, a greyish white vascular lesion causingopening of the Sylvian fissure and spreading along either sideof the sphenoid ridge into both the temporal and frontal areaswas seen. The mass also infiltrated the ipsilateral branches ofthe internal cerebral artery, the middle cerebral artery, and theoptic nerve. A provisional squash (cytology) performed intra-operatively from the mass showed lymphocytes, plasma cells,and foreign-body giant cells infiltrating collagenous tissue withnumerous fungal filaments. These features were reported to beconsistent with a fungal infection. A total excision of the masswas performed, and the mass was subjected to histological andmicrobiological analysis.

Histology. The material submitted for histology consisted ofmultiple grey-white firm tissue bits with areas of hemorrhages.Multiple sections from the specimen stained with hematoxylinand eosin revealed fragments of collagenous tissue infiltratedby lymphocytes and plasma cells along with numerous multinu-cleate giant cells. There were extensive areas of necrotic fociconsisting of thin, short, and irregular fungal filaments distrib-uted sparsely. Native tissue could not be identified. The mor-phology of these filamentous structures resembled those ofAspergillus hyphae on Gomori methenamine silver stain (Fig.2). The mass was reported as being suggestive of cerebral

* Corresponding author. Mailing address: Department of Microbi-ology, Nizam’s Institute of Medical Sciences, Punjagutta, Hyderabad-500 082, Andhra Pradesh, India. Phone: 91 040 425 6478. Fax: 91 040331 0076. E-mail: lgorthi@hotmail.com.

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aspergillosis. The fungal filaments did not show the presence ofmelanin pigment in their cell walls.

Microbiology. A direct microscopic examination and KOHmount of the tissue revealed septate, dichotomously branchinghyphae, and the specimen was interpreted as probably belong-ing to the genus Aspergillus. The specimen was inoculated onto(i) two slopes of Sabouraud’s dextrose agar (SDA) with anti-microbial agents chloramphenicol and cycloheximide; (ii) twoslopes of SDA without antimicrobials; and (iii) a slope of brainheart infusion agar (BHI). The SDA slopes were incubated at28 and 37°C, while the BHI slope was kept at 37°C. Based onthe histology and direct microscopy reports, the patient begana regimen of amphotericin B injection. He tolerated the injec-tion well. There were no postoperative complications.

A postoperative CT scan showed only a minute residue ofring enhancement of 1.5 to 2 mm (Fig. 3). At the time ofdischarge, the patient was experiencing persistent nerve palsyinvolving the right second, third, fourth, fifth, and sixth cranialnerves. The patient was requested to continue following anamphotericin infusion protocol with 50 mg (0.6 mg/kg of bodyweight/day) of amphotericin B, under a physician’s care. Atotal cumulative dose of amphotericin up to 1.5 to 2.0 g wasadvised. The patient continues to report to the neurosurgeryoutpatient clinic for review every 4 weeks and has shown steadyimprovement in the 1.5 years of follow-up.

Mycological examination. After 3 days of incubation, growthof a brown filamentous fungus was observed on all the SDAand BHI slopes.

The morphology of the branching conidiophores with single-cell conidia with acuminate base did not correspond to thegenus Aspergillus or any commonly known dematiaceous fun-gus. For definitive identification of the fungal isolate, wesought the expert assistance of D. Swinne, Head of the Mycol-ogy Division, Prince Leopold Institute of Tropical Medicine,Antwerp, Belgium. The isolate was forwarded to H. Beguin atthe Scientific Institute of Public Health, IHEM Culture Col-lection, Brussels, Belgium. After a thorough and meticulousexamination, H. Beguin identified the isolate as belonging tothe mitosporic genus Nodulisporium.

The fungus was determined to be dematiaceous as it wasdark in color.

Cultures grew readily on any of the usual laboratory culturemedia. On 2% malt agar and oatmeal agar, at 25°C, the funguscovered a 9-cm-diameter plastic petri dish within 1 week.Growth at 37°C was fast, reaching a diameter of 80 mm in 7days. Growth at 40°C was slow, reaching only 5 mm after 1week. The culture did not grow at 45°C.

The colony was plane and velutinous, with irregular marginsand mycelium subsurface. The conidiogenous areas were scat-tered over the entire surface of the colony and were palebrownish, near Isabelline (color code, R. 65 [reference 18]).Stromata, exudates, and soluble pigment were absent. Thereverse was a deeply colored dark brown, near dark mousegrey (color code, R. 119 [reference 18]).

The mycelium was composed of septate hyphae, about 2 to3 (5.5) �m (extreme value is shown in parentheses) in diame-

FIG. 1. (Left) CT scan of the brain showing the large, irregularly shaped, slightly hyperdense enhancing lesion in the right medial temporo-parietal region, extending to the orbit and the paranasal sinuses, with surrounding edema. (Right) CT scan of the brain at another level showingclearly the mass effect produced by the lesion, with shift of the midline structures to the left.

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ter, and slightly roughened. The conidiophores were erect,mononematous (coindiophores solitary), irregularly branched,flexuous (wavy, not rigid), septate, and becoming mid-brown inthe apical part with age. In general the conidiophores were 120to 200 �m tall, 2.2 to 3.3 �m wide, and smooth, or, like themycelium, they were slightly roughened and often studded withdark granules. Conidiogenous cells were seen arising, singly ormore often in groups, laterally or more frequently at eachbranch terminus. They were elongate-clavate, mostly 9.0 to30.0 �m by 2.2 to 3.3 �m, with denticles closely crowded at thetip of the cells that became more or less slightly swollen fromrepeated conidial production (Fig. 4).

Conidia arose singly and successively on denticles at the tipsof conidiogenous cells; the first conidium was formed apically.Subsequent conidia were formed sympodially in more or lessbasipetal succession, forming heads. Conidia were pale brownand smooth, 5.5 to 7.7 �m (12) by 3.3 to 4.4 (5.2) �m, dry,single celled, and ellipsoidal, with a flattened base indicatingthe former point of attachment to conidiogenous cell. Therewere no apparent morphological differences in the conidiog-enous structures from colonies growing on oatmeal agar(Difco).

The conidium production on areas of the colony surface, theconidiophores, and conidiogenesis were similar to those occur-ring in many anamorphs of Hypoxylon Bull; more specifically

they were similar to those occurring in the genus Nodulispo-rium.

Discussion. Among the subdivisions of infections of the cen-tral nervous systems (CNS) the two following infections aredue to dematiaceous species: (i) rhinocerebral phaeohyphomy-cosis, a typical secondary infection with airborne conidia whichcan germinate in the sinus and grow into the brain (the cere-bral involvement is typically secondary), and (ii) cerebral phae-ohyphomycosis, a primary infection by a fungus located exclu-sively in brain parenchyma, with the first symptoms being ofcerebral origin (14). In the latter case lungs are supposed to bethe primary site of infection, from where the fungus spreads viablood to the brain.

The main etiologic agents of infections of the CNS areCladophialophora bantiana (Sacc) de Hoog et al., Ramichlo-ridium mackenziei Campbell et Al-Hedaithy, Exophiala derma-titidis (Kano) de Hoog, and Ochroconis gallopava (W. B.Cooke) de Hoog (2, 8, 9, 13, 17). Rhinocladiella atrovirensNannfeldt also has been reported as a neurotropic pathogen(7). Among these agents of phaeohyphomycosis, only the gen-era Rhinocladiella and Ramichloridium form single-celledconidia by sympodial growth on a pale brown rachis withcrowded conidium-bearing denticles like the fungus isolatedfrom the present case. However, the isolated fungus has erectand branched conidiophores whereas these structures are ab-

FIG. 2. Thin, irregular, short hyphae of Nodulisporium sparsely distributed in tissue. Shown is a section of brain tissue stained with Gomorimethenamine silver stain. Magnification, �350.

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sent or little differentiated from the vegetative hyphae in Rhi-nocladiella. Conidiophores in Ramichloridium are unbranched.Furthermore, R. mackenziei is limited to the Middle East (14).

Among the genera with acropleurogenous conidia are alsoGeniculosporium Chesters et Greenhalgh, Dicyma Boulanger,Dematophora Hartig, and Nodulisporium Preuss (11). The ge-nus Geniculosporium has conidiogenous cells, usually with along geniculate rachis. In 1981, von Arx (23) transferred Hans-fordia Hughes, Basifimbria Subramanian et Lodha, Gonytrich-ella Emotto et Tubaki, and Puciola de Bertoli to the genusDicyma, which has been redescribed as having smoothbranched conidiophores, often partly forming septae, whip-likehyphae or cylindrical-clavate, ampulliform cells (24). Demato-phora, like Geniculosporium, has sympodulosporous conidiabut forms conidia in two rows in the apical fertile parts andsynnemata in nature, unlike in cultures (25). Finally, the formgenus Nodulisporium that has swollen conidiogenous areas(conidiogenous cells denticulate, cylindrical to clavate) corre-sponds to the isolated strain. Moreover, branching patterns ofconidiogenous structures, i.e., profusely branched conidio-phores and the tendency to produce more than two conidiog-enous cells at each branch terminus, are also referable toNodulisporium (15).

Nodulisporium was erected on the basis of two moniliaceousspecies: Nodulisporium album Preuss and Nodulisporiumochraceum Preuss. Subramanian restricted Nodulisporium topale colored species and placed the dematiaceous species inthe genus Acrostaphylus Arnaud ex Subramanian (22). Green-halgh and Chesters (12) do not accept this division, based on

pigmentation. Indeed, many members of the Xylariaceae familyare hyaline at first and colored later on with age. Regardless ofthis, Subramanian from India related two Acrostaphylus spe-cies: Acrostaphylus hyperparasiticus Subram, with globoseconidia, and Acrostaphylus lignicola Subram, with conidia ovalto elliptical up to 7 �m long (22). These two taxa do notcorrespond to the present isolate. Later, two other species, alsooriginating from India, were described as Nodulisporium:Nodulisporium indicum Reddy et Bilgrami (only related twoAcrostaphylus species represented by the type culture isolatedin 1967 from Mangifera indica, but this culture shows manychlamydospores and arthroconidia, absent in the case strain)and Nodulisporium griseobrunneum Mehrotra (isolated fromsoil in a Piper betel orchard). The latter species only differsfrom the present isolate in having slightly larger (6.6 to 13 �mby 3.5 to 5.5 �m) conidia; but the ability to grow at 37°C, thegrowth rate, the colony color, and the conidiophore morphol-ogy closely resemble those of N. griseobrunneum.

The fact that this strain isolated from brain tissues growsvery rapidly at 37°C is not a common occurrence. This char-acteristic should not be ignored or overlooked in the protologof the name of a species. The literature only refers to N.cylindroconium de Hoog (4), of which the type culture hadbeen isolated as Tritirachium species by Evans (10, 11) as astrong thermotolerant species with an optimal growing tem-perature at 40°C (growth to a diameter of 35 mm within 10days on malt agar) (14). The other members of the genusNodulisporium also differ distinctly by the size of the conidia(mostly smaller), by the branching habit, and by the appear-ance of the colonies (4, 5, 6, 10, 19, 20, 21, 26).

Nodulisporium is an anamorph also associated in nature withmany ascomycete species of the Xylariaceae family, especiallyHypoxylon. Ju and Rogers (16) even consider this anamorph asa primary criterion for recognizing a xylariaceous fungus as amember of the genus Hypoxylon. Among these ascomycetes,some produce conidia on areas of the entire surface of thecolony. This feature occurs in the strain isolated in the presentstudy and it is more characteristic in the Hypoxylon species (1).A few members of this genus are strictly host limited on varioustrees; others are found on dead branches or fallen trunks andlogs, often accompanied or preceded by a Nodulisporium state.Following the International Code of Botanical Nomenclature(article 59), the legitimate name of a holomorphic species canbe typified by only its teleomorph, i.e., the morph characterizedby ascospores in ascomycetous fungi (12a). This situation isparticularly true in xylariaceous fungi for which dual nomen-clature exists, although not used often, and species are mostlydescribed only under the ascomycetous name. Indeed theNodulisporium state frequently disappears early in the processof stromal development (12). This implies that most keys arebased on teleomorph features only, the anamorphic state beingignored or little described—as “a Nodulisporium-like ana-morph,” as “a typical Nodulisporium,” or as referable to thisgenus by the use of “Nodulisporium state of . . ..” In culture,ascigerous stromata are absent, but some of these ascomycetesproduce conidia, and then the identification of a Xylariaceaeanamorph should be compared with the asexual states thatoriginated from identified teleomorphic material.

The identification of this anamorphic isolate to the species

FIG. 3. Postoperative CT scan showing near-total excision of thetumor.

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level remains problematic in spite of its unusual rapid growthat 37°C, which is an uncommon characteristic.

The brain abscess was the only conspicuous pathology in thiscase report. There were no sinusitis, no underlying diseaseprior to the infection, and no lesion outside the CNS. Theportal of entry of infection has thus not been identified. Per H.Beguin, this is the first case of cerebral infection by Noduli-sporium and the second case involving this fungal taxon inhuman disease. Also, Nodulisporium is for the first time beingdescribed here as a pathogenic agent of human infection. Coxet al. reported the first case of an allergic fungal sinusitis by thepresence of a persistent Nodulisporium species in mucus in1994 (3). However, there was no tissue invasion by the fungusin their study.

Our isolate is preserved at the BCCM/IHEM Culture Col-lection (IHEM 16563).

We sincerely appreciate the interest and efforts taken by H. Beguinin identifying the fungal isolate and providing the relevant literature.

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FIG. 4. Microscopic morphology of Nodulisporium. (A) Unstained wet mount showing the dematiaceous nature of the fungus; (B) Lactophenolcotton blue mount of the fungus showing slightly roughened, erect, irregularly branched conidiophores, with denticles closely crowded at theswollen tips of the conidiogenous cells and conidia. Magnification (both panels), �300.

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