JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2010, p. 251–257 Vol. 48, No. 10095-1137/10/$12.00 doi:10.1128/JCM.00018-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Phenotypic and Molecular Characterization ofMadurella pseudomycetomatis sp. nov., a

Novel Opportunistic Fungus PossiblyCausing Black-Grain Mycetoma�

Jie Yan, Jun Deng, Cun-Jian Zhou, Bai-Yu Zhong, and Fei Hao*Department of Dermatology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China

Received 5 January 2009/Returned for modification 18 March 2009/Accepted 9 November 2009

A case of black-grain mycetoma occurring on the lower jaw with an odontogenic origin, which to ourknowledge is the first case reported in China, is presented here. The clinical manifestation, histopathologicalmorphology, and microbiological features are described. The new species, Madurella pseudomycetomatis, iso-lated from the black grains discharged by this patient, was analyzed using sequence data of the multiloci ofribosomal DNA (rDNA) and its ability to ferment carbohydrate as well as morphology. The analyses of theinternal transcribed spacer (ITS) region and the D1/D2 hypervariable region of the 28S ribosomal genesequences support a new species designation. Antifungal susceptibility testing was conducted, indicating thatMadurella pseudomycetomatis was highly susceptible to itraconazole, voriconazole, and amphotericin B; mod-erately susceptible to terbinafine; and resistant to fluconazole and flucytosine.

Mycetoma is a chronic, granulomatous, inflammatory dis-ease and is characterized by the triad of a tumefaction, multi-ple draining sinuses, and the presence of grains caused by truefungi (eumycetoma) or filamentous bacteria (actinomycetoma)(4, 15). The disease is endemic in tropical and subtropicalareas. It is predominately seen in India, Africa, and SouthAmerica, while rarely encountered in Europe. However, withincreasing numbers of immigrants and tourists, mycetoma isfrequently imported into Western countries (3, 11), although itseldom occurs in China. Between 1960 and 2008, there hadbeen only 18 cases reported in China, of which 9 were eumy-cetoma and 9 were actinomycetoma. The etiological agentswere various, including Nocardia brasiliensis, Nocardia aster-oides, Nocardia otitidiscaviarum, Actinomadura madurae, Acre-monium falciforme, Scopulariopsis maduromycosis, Pseudall-escheria boydii, Madurella mycetomatis, Trichophyton verrucosum,and Aspergillus.

Mycetoma usually affects adult male laborers who workbarefoot in rural areas. The most commonly affected site is thefoot (70%); however, other exposed body parts such as thehand, leg, knee, arm, thigh, and perineum can be infectedoccasionally. Rarer sites on the paranasal sinuses, mandible,intraspine, bladder, brain, and lung have been reported (4, 15).Craniofacial mycetoma is extremely rare, especially that causedby fungi, and is the most difficult form to treat. Gumma et al.(16) showed that mycetoma involving the head and neck ac-counted for 15 of 400 cases (3.75%). An investigation by Lynch(19) indicated that the rate of the cranial infection was only 3of 317 cases in eumycetoma and 15 of 233 cases in actinomy-

cetoma: i.e., 15 out of 18 mycetoma infections of the head weredue to actinomycetes.

Here we present an extraordinary case of craniofacial eumy-cetoma extending from gum to lower jaw in a 27-year-oldChinese male. The case is worth reporting not only by itsrareness in China but also its unusual affected site. Moreover,we have isolated a distinctive dematiaceous fungus from clin-ical specimens from this patient. By sequencing of internaltranscribed spacer 1 (ITS1)-ITS2 region, it has maximum se-quence identity (93%) with Madurella mycetomatis, one of themain microorganisms causing black-grain fungal mycetoma.Further morphological, physiological, and molecular studiesdemonstrated that the isolate belonged to an as-yet-unre-ported species. The taxon is fully described, illustrated, andanalyzed in the work below and has been proposed as a newspecies of Madurella.

CASE REPORTS

A 27-year-old man, originating from Chongqing, China, wasfirst seen at Southwest Hospital on 2 February 2006. The pa-tient presented with a swelling on his lower jaw, showing mul-tiple fistulae which discharged black grains. He had a history ofseveral nodules on the middle gum 6 years ago. In the earliestobserved stage, the nodule was about a pea in size and suc-ceeded by some small blebs. When the blebs ruptured, multiplesinuses formed. The patient was mistakenly diagnosed with“tumor of root tip of the tooth” and received an extraction ofthe central incisor and partial alveolectomy in October 2002.At that time, he received cotrimoxazole and penicillin for sev-eral months without apparent improvement. The abscess grad-ually spread to the lower jaw and quickly formed multipledraining fistulae, discharging pus and black grains. The patientdid not recall any trauma or puncture at the site of his lesion.The route of entry of the organism was undetermined. Onphysical examination, a large swelling measuring 8 by 6 cm on

* Corresponding author. Mailing address: Department of Derma-tology, Southwest Hospital, Third Military Medical University, Chong-qing, China. Phone: 86 23 68754416. Fax: 86 23 65462522. E-mail:haofei62@medmail.com.cn.

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the lower jaw was seen, which was hard and woody to thetouch. Three protuberant sinuses were observed, extendingfrom the lower jaw to the floor of the mouth, with some ofthem discharging black granules (Fig. 1A). Intraorally, thelower teeth from left canine to right canine were absent, andthere were multiple sinuses in his mouth floor and middle gum(Fig. 1C). The patient looked healthy with normal systems, buthad a 20-year history of infection with B-type hepatitis (HB).Blood tests revealed impaired liver function. HBs antigen,HBe antigen, and HBc antibody were positive. HBV DNAPCR showed viral proliferation. The patient underwent a com-puted tomography scan, showing numerous heterogeneous softtissue masses with an osteolytic lesion in the mandible body(Fig. 2A). Granulomatous inflammation was confirmed his-topathologically. Periodic acid-Schiff (PAS) staining showedthat a fungal grain was embedded in granulation tissue, andnumerous broadly branched, separate hyphae approximately 5�m in diameter grew toward the periphery of the grain (Fig.2B). One week after admission to the hospital, the patientunderwent a surgical debridement in which massive soft tissuescontaining multifocal abscesses and burrowing sinus tractswere excised. Numerous hard black grains with irregular sizeand shape were seen in the surgical specimens. Microscopicallyin KOH mounts, the grains were composed of many dematia-ceous hyphae with some brown, swollen cells (Fig. 2C). Afterbeing washed with sterile saline, the grains were seeded ontoSabouraud’s dextrose agar (SDA) and incubated at 25 and37°C. After 3 weeks of incubation, several slow-growing, cau-liflower-like, granular colonies producing brown, diffusing pig-ments were obtained (Fig. 2D). No other organisms weregrown from the specimen. A diagnosis of eumycetoma wasmade, but the isolated fungus could not be adequately identi-fied by classical mycology. The strain was tentatively coded as“TMMU3956.” The patient was managed with several surgicaldebridements and received intravenous amphotericin B for 4weeks, succeeded by oral itraconazole for 12 months accordingto routine protocol. In the beginning, a satisfactory response to

treatment was achieved. The sinuses almost closed and thesubcutaneous swelling dramatically decreased (Fig. 1B and D).The patient became mobile and even worked as a salesmanemployed by a company. Over time, however, in May 2008 thewound began to drain again, possibly due to the fact that theexcision was incomplete or the patient didn’t insist on treat-ment because of the costliness of the azole drugs.

MATERIALS AND METHODS

Morphological and physiological studies. The isolate was inoculated ontoplates of Sabouraud’s dextrose agar (SDA), cornmeal agar (CMA), potato dex-trose agar (PDA), Czapek Dox agar (CDA), and brain heart infusion agar(BHIA) (Sigma-Aldrich) as well as standard slide culture preparations andincubated at 37°C for 8 weeks. Both gross morphological and microscopic ob-servations were made. In addition, scanning electron microscopy was used tostudy morphological growth characteristics. To obtain the maximum growthtemperature, the strain was subcultured onto PDA slants and incubated at 25°C,37°C, 40°C, 42°C, and 44°C for up to 4 weeks. The ability of the isolate toassimilate a carbohydrate source was determined with the API 20C AUX system(bioMerieux, Marcy l’Etoile, France) (14). Prior to the carbohydrate assimilationtest, the homogenized fungal suspension was prepared by ultrasonic treatment aspublished previously (7) and then was standardized to a 2 McFarland standardwith the medium provided. Finally, 100 �l of this inoculum was used to fill thecupules of the test strips as directed by the manufacturer.

rRNA gene sequencing. DNA extraction was performed by the glass beads–salting-out procedure (21). The 18S region was amplified by PCR with primersNS1, NS2, NS3, NS4, NS5, NS6, NS7, and NS8 (26). The ITS1-ITS2 region andD1/D2 hypervariable region of the 28S rRNA gene were amplified by PCR withprimers ITS5 and ITS4 (2) and NL1 and NL4 (22), respectively. Similarly, PCRamplification of the ITS1 region and ITS2 region was performed using primersITS1 and ITS2 (9) and ITS3 and ITS4 (8), respectively. In addition, M. myce-tomatis-specific PCR for species identification using primers 26.1A and 28.3Awas performed (2). The PCR products were visualized on an agarose gel afterethidium bromide staining and were sequenced commercially (Sangon, China)after purification. A BLAST search for each sequence was performed to identifybest matches.

Phylogenetic analysis. For phylogenetic analysis, the GenBank sequences in-dicated in Fig. 7 were used. This database was made on the basis of the knownspectrum of filamentous fungi responsible for eumycetoma in PubMed. Phylo-

FIG. 1. (A and C) Clinical appearance before treatment. Note thelarge swelling on the lower jaw with some sinuses extending from lowerjaw to mouth floor. (B and D) Clinical appearance after 1 month oftreatment. Note the decreased swelling and the nearly closed sinuses.

FIG. 2. (A) Computed tomography scan showing numerous heter-ogeneous soft tissue masses with an osteolytic lesion in the mandiblebody. (B) Fungal hyphae growing toward the periphery of the grain.PAS staining. Magnification, �200. (C) Fungal grains seen microscop-ically in KOH mounts. The grains were composed of many dematia-ceous hyphae with some brown, swollen cells. (D) Twenty-one-day-oldculture of the black grains on SDA. The colony is slow-growing, gran-ular, and cauliflower-like, producing brown, diffusing pigments.

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genetic trees of ITS1-ITS2 were constructed by the neighbor-joining methodusing the MEGA 4.1 software with a gap opening penalty value of 15 and adefault gap extension penalty value of 6.66. Confidence values for individualbranches were determined by bootstrap analyses (1,000 replicates).

Antifungal susceptibility. The MICs of fluconazole, flucytosine, itraconazole,amphotericin B (Sigma-Aldrich), terbinafine (Novartis Pharma Ltd., Beijing,China), and voriconazole (Pfizer Pharmaceuticals Limited, Dalian, China) weredetermined by the modified CLSI (formerly NCCLS) broth microdilutionM38-A method (20). The homogenized fungal suspension without conidia wasprepared by the method of Ahmed et al. (7). Finally, 100 �l of this 2-fold-dilutedinoculum (approximately 2 � 104CFU/ml) was applied to the 96-well microplate.

Experimental model in animals infected with strain TMMU3956. In the ex-periment, the inoculum containing 107 CFU per ml in saline was prepared asdescribed above. Eight-week-old BALB/c mice weighing 20 g and 3-month-oldNew Zealand White rabbits weighing 2 kg (male/female) were used. At first, theBALB/c mice and New Zealand White rabbits were divided into two groups (Fig.3). In one group, 50 mg of cyclophosphamide (Chongqing, China) per kg of bodyweight per day (on days 1, 3, 5, 7, and 9) was injected intraperitoneally to induceinitial immunosuppression. Subsequently, these mice and New Zealand Whiterabbits (immunocompetent/immunocompromised) were divided into four sub-groups. In subgroup I, a suspension of mycelia at 5 ml (107 CFU/ml) per kg ofbody weight was injected intravenously on day 9. In subgroups II and III, asuspension of mycelia at 5 ml per kg of body weight was injected intraperitoneallyand subcutaneously, respectively. For comparison, in subgroup IV (control), theanimals were injected with physiological saline. Reimmunosuppression and re-inoculation was performed at 4-week intervals. The infected mice and NewZealand White rabbits as well as those uninfected were monitored closely everyweek and euthanized on days 28, 56, and 84 and checked for the presence of thecharacteristic black grains and detectable infection in their subcutaneous tissue,peritoneum, and internal organs. The specimens from the muscle, lung, liver,spleen, and kidney were subjected to histopathological examination, and thehomogenized organs were cultured for fungus TMMU3956.

RESULTS

Morphological and physiological studies. The growth rate isslow and the colonies’ morphology varied on different media.Among the total, the colonies on BHIA grew best. They wereround, flat, downy, and about 15 mm in diameter, with actino-morphous reductus, producing a brownish diffusible pigment(Fig. 4A). However, the colonies on SDA, PDA, CDA, andCMA were very different from those on BHIA. They weregranular, raised, and cauliflower-like and measured 3 to 8 mm

FIG. 3. Experimental protocols. The BALB/c mice and New Zealand White rabbits were divided into two groups. To lower immunologicalcompetence, 50 mg of cyclophosphamide per kg of body weight per day (on days 1, 3, 5, 7 and 9) was injected in group 2. Next, each group wasdivided into four subgroups. In subgroup I, a suspension of mycelia at 5 ml (107 CFU/ml) per kg of body weight was injected intravenously on day9. In subgroups II and III, the same inocula were injected intraperitoneally and subcutaneously, respectively. In subgroup IV (control), the animalswere injected with physiological saline. Reimmunosuppression and reinoculation were performed at 4-week intervals.

FIG. 4. (A) Colonies on BHIA after a 3-week culture. The coloniesare round, downy, and 15 mm in diameter, with actinomorphous re-ductus, producing a brownish diffusible pigment. (B) Colonies on PDAafter a 3-week culture. The colonies are granular, raised, cauliflower-like, and 3 to 8 mm in diameter. (C) Microscopic examination of the4-week-old slide culture on SDA. Numerous chlamydospores, interca-lary or terminal, are detected. (D) Microscopic examination of the4-week-old slide culture on CDA. Septate hyphae devoid of spores,conidia, and chlamydospores are detected. (E) Microscopic examina-tion of the 4-week-old slide culture on CMA. Many sclerotia at theapex of hyphae are detected. (F) Electron microscopic appearance ofthe phialospores. The conidia in short phialide are smooth, ellipsoidal,or spherical and 1 �m in diameter. Magnification, �3,600.

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in diameter (Fig. 4B). Microscopically, numerous chlamyd-ospores, which were subglobose, thick-walled, 15 �m in diam-eter, and intercalary or terminal, were observed on SDA andPDA (Fig. 4C), while the fungus remained sterile on BHIAand CDA (Fig. 4D). On CMA, many sclerotia at the apex ofhyphae were detected (Fig. 4E). Electron microscopic appear-ance revealed the means of asexual multiplication of strainTMMU3956, the phialospores. The solitary blastoconidium inphialide was smooth, ellipsoidal, or spherical and approxi-mately 1 �m in diameter (Fig. 4F), while the hyphae wereslender, 2 to 5 �m in diameter, with apparent septa andbranching. The fungus showed optimal growth at 37°C, could

tolerate 42°C, but stopped growth at 44°C. Assimilation ofglucose, arabinose, xylose, cellose, maltose, and trehalose waspositive in the API 20C AUX system.

rRNA gene sequencing. The amplified DNA fragments ofthe 18S region were 551 bp, 601 bp, 307 bp, and 375 bp (Fig.5A). The complete length of 18S region was 1,767 bp aftersplicing. A search of the GenBank database for the sequencerevealed a high level of similarity to the sequence of Madurellamycetomatis (100% similarity). Its GenBank accession numberwas EU815932. PCR using primers ITS5 and ITS4 yielded a608-bp amplicon (Fig. 5A). The closest match in the GenBankdatabase was Madurella mycetomatis, with 93% similarity. ItsGenBank accession number was EU815933. The sequences ofthe three ribosomal regions (ITS1, ITS2, and D1/D2) were 257bp, 347 bp, and 602 bp, respectively (Fig. 5B). For the ITS1 andITS2 regions, the closest sequence was Madurella mycetomatis,with 89% and 96% similarity, respectively. The D1/D2 regionshowed a sequence similarity of 98% with an uncultured AMFfungus because no data for the D1/D2 region of Madurellamycetomatis were available. Their GenBank accession num-bers were EF600937, EF600938, and EF600939. M. mycetoma-tis-specific primers didn’t amplify the DNA extracted fromstrain TMMU 3956. The results of the BLAST search indi-cated that primers 26.1A and 28.3A had poor similarity to thehomologous region of strain TMMU3956 (Fig. 6).

Phylogenetic analysis. In the ITS1-ITS2 phylogenetic tree(Fig. 7), two main clusters were distinguished. The first clusterincluded relevant clinical species of Aspergillus, Microsporum,Trichophyton, Exophiala, Leptosphaeria, Corynespora, Pyrenochaeta,Curvularia, Bipolaris, and Madurella grisea, which were selectedfor comparative purposes. The second cluster included twomain subclusters, also containing some species which wererelatively distant from other sequences, such as Curvularia

FIG. 5. (A) Lane 1 contains a 100-bp molecular size marker. Lanes2 to 5 show the PCR products amplified by NS1 and NS2, NS3 andNS4, NS5 and NS6, and NS7 and NS8, respectively. Lane 6 shows theamplicon of ITS1-ITS2 using the primers ITS5 and ITS4. (B) Lanes 1and 5 contain a 100-bp molecular size marker. Lanes 2 to 4 show thePCR products amplified by ITS1 and ITS2, ITS3 and ITS4, and NL1and NL4, respectively.

FIG. 6. Homologous comparison of the ITS1-ITS2 region between M. pseudomycetomatis (sequence 1; EU815933) and M. mycetomatis(sequence 2; AF162133). Primers 26.1A and 28.3A based on the M. mycetomatis ITS1-ITS2 region involved 71 to �90 bp and 494 to �474 bp,respectively, showing poor similarity to the homologous region of M. pseudomycetomatis. Color highlighting emphasizes the similarity of anddiversity between sequence 1 and sequence 2.

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pallescens and three species of Phialophora. Strain TMMU3956and the type strain of M. mycetomatis, which formed a terminalbranch with 100% bootstrap support, together with Fusarium,Acremonium, and Cylindrocarpon constituted a subcluster. Theother subcluster was formed by the rest of the fungi: i.e.,Scopulariopsis, Polycytella, Phialophora, Pseudallescheria, andCephalosporium.

Antifungal susceptibility testing. Strain TMMU3956showed high susceptibilities to itraconazole (MIC-0 � 0.0625�g/ml), voriconazole (MIC-0 � 0.0313 �g/ml), and amphoter-icin B (100% growth inhibition [MIC-0] � 0.0313 �g/ml), whileit was resistant to fluconazole (�50% growth inhibition [MIC-2] � 32 �g/ml) and flucytosine (MIC-2 � 64 �g/ml). The strainwas less susceptible to terbinafine. The MIC-0 was 2 �g/ml.

Experimental model in animals with strain TMMU3956.In all groups, the survival rate of BALB/c mice and NewZealand White rabbits was 100%. No characteristic blackgrains or detectable infection were observed. Pathologicalspecimens from the tissue or viscera didn’t show any

changes caused by fungal proliferation. The cultures of theorgans were negative.

DISCUSSION

Since mycetoma is classified into eumycetoma and actinomy-cetoma, identification of these two different types is important todevelop an appropriate plan of treatment. In general, eumyce-toma has a slow course compared to actinomycetoma. The si-nuses in eumycetoma tend to be proliferative and protuberant,while in actinomycetoma, the sinuses are flat or depressed. His-topathologically, eumycetomas with fungal grains characterizedby broad mycelial filaments are readily differentiated from acti-nomycetomas with fine filaments. Moreover, black-grain myceto-mas are only caused by fungi (15, 19). Thus, our patient wascorrectly diagnosed from the clinical manifestation, emission ofblack grains, and histopathologic findings as being infected witheumycetoma. However, the causative fungi responsible for eumy-cetoma are diverse, and most of these fungi are described as

FIG. 7. Distance tree of ITS1-ITS2 rRNA gene sequences of 40 species responsible for eumycetoma. The tree was constructed using theneighbor-joining method with MEGA 4.1 software.

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nonsporulating agents because of their poor or delayed sporula-tion. Identification of these fungi by standard mycological proce-dures challenges the medical microbiological laboratory.

Fortunately, the development of DNA-based molecular ap-proaches significantly improved the sensitivity and specificity indetection of etiological organisms. Especially, the systematicanalyses of ITS1, ITS2, and D1/D2 hypervariable region havebeen proven to be effective in identifying the pathogenic moldsto the species and sequevar levels, while ribosomal genes in-cluding 18S and 28S genes (the 26S gene in all yeasts), whichare relatively conserved, provide useful phylogenetic informa-tion. Strains with �1% sequence diversity in the D1/D2 do-main or ITS region usually represent separate species (8, 9,22). Therefore, based on PCR and sequencing of the ribosomalDNA (rDNA), the results confirmed that the causative agentof our patient belonged to the genus Madurella by its 100%similarity to the sequence of M. mycetomatis at the 18S locus.Moreover, from the phylogenetic tree based on the ITS1-ITS2rRNA gene sequences, we can see that strain TMMU3956 andM. mycetomatis could be aligned into a cluster with 100%confidence, with the genera Fusarium and Acremonium, whichbelong to the class Pyrenomycetes of Ascomycetes, as its nearestneighbors. However, M. grisea, the other member of Madurella,is closer to the genera Leptosphaeria, Curvularia, and Bipolaris,which belong to the class Loculoascomycetes of Ascomycetesrather than to the class Pyrenomycetes. These data were inaccord with the known publications on the matter (12, 13),which demonstrated that M. mycetomatis and M. grisea shouldbelong to different orders of Ascomycetes, viz. M. mycetomatisbelongs to the order Sordariales and M. grisea is likely to be amember of the order Pleosporales. However, although strainTMMU3956 has the nearest distance to M. mycetomatis, PCRwith M. mycetomatis-specific primers couldn’t amplify thegenomic DNA of strain TMMU3956. The significant sequencediversity at the ITS1 and ITS2 loci compared with M. myce-tomatis elicited the conjecture that it probably belonged toanother species closely related to M. mycetomatis.

It is well known that Madurella species are the most com-monly reported agents causing black-grain mycetoma. Twospecies, M. mycetomatis and M. grisea, are recognized. From1999, many studies focused on the genetic variability in M.mycetomatis by using restriction endonuclease assay (REA),random amplified polymorphic DNA(RAPD), restriction frag-ment length polymorphism (RFLP), amplified fragment lengthpolymorphism (AFLP), and sequencing of the rDNA smallsubunit (SSU) and ITS (1, 2, 5, 12, 13, 17, 24). It is acceptedthat the M. mycetomatis strains obtained from Sudan are ho-mogenous, whereas those from other countries are somewhatheterogeneous (1, 2, 5, 12, 13, 17, 24). What is more, a numberof investigators found that a cluster of strains initially identifiedas M. mycetomatis showed DNA fingerprints completely dif-ferent from that of the type strain (1, 12, 13, 24). All of thesestrains had more than 5% ITS diversity from M. mycetomatis(approximately 39 bp involved), and none of them had anAfrican origin, raising doubts about the species status of theseisolates. One explanation is that the sequence variability at ITSlocus of “M. mycetomatis” might be related to the geographicalarea and the climatic environment. The other, more likely,explanation is that these strains represent a separate speciesand the outdated taxonomy of Madurella needs improvement.

To demonstrate strain TMMU3956 indeed represents anovel species different from M. mycetomatis and M. grisea, acombination of morphological and physiological tests was con-ducted. Morphologically, strain TMMU3956 is more similar toM. mycetomatis than M. grisea (4, 25), but in most cases,TMMU3956 formed granularis rather than woolly coloniesand grew most slowly. In view of the wide polymorphism whichMadurella species often showed, species differentiation of Ma-durella can be complemented by differences in sugar assimila-tion and optimal growth temperature (4, 14). M. mycetomatisgrows well at 37°C and assimilates lactose but not sucrose,whereas M. grisea stops growth at 37°C and assimilates sucrosebut not lactose (4, 25). Our results indicated that strainTMMU3956 could tolerate 42°C but assimilates neither su-crose nor lactose. Their distinctive physiological patterns notonly provided great help in distinguishing these species butalso gave a cogent argument for supporting the hypothesis thatstrain TMMU3956 represents a novel species of Madurella.

It is important to identify the causative fungi to the specieslevel because different species probably have different suscep-tibilities to antifungal agents. There are only a few reportsabout the in vitro susceptibility of the fungus Madurella myce-tomatis (7, 23), indicating that the antifungal activities ofketoconazole, itraconazole, and voriconazole were superior tothose of fluconazole, flucytosine, and amphotericin B. For itra-conazole and voriconazole, our result was in good agreementwith these earlier studies. However, for amphotericin B, whichhad been demonstrated to be less effective on M. mycetomatis,our results dissented from their findings. A high susceptibilitywas obtained that was as effective as that of itraconazole andvoriconazole. In addition, a susceptibility difference (2 �g/mlversus 0.015 �g/ml) to terbinafine between strain TMMU3956and M. mycetomatis was observed (18).

The production of eumycetoma in animals under laboratoryconditions is difficult. Although some investigators have beensuccessful in developing M. mycetomatis infection in an animalmodel (6), reproducibility was poor. The determinants impor-tant for the establishment of eumycetoma in experimental an-imals are not yet elucidated. It is accepted that successfulinfections are usually inoculum dependent and require acaciathorns, killed tubercle bacilli, Freund’s incomplete adjuvant, orsterilized soil from the endemic region as adjuvants. However,many current studies have demonstrated that adjuvant addi-tion is not required for the production of infection (10, 27). Inour experiment, we attempted to use a fungal suspension with-out any adjuvant as a natural inoculum. Unfortunately, neitherblack grains nor local tumor formation was observed, althoughdifferent routes of inoculation and host immune status wereattempted. It is likely that the adjuvants are absolutely neces-sary for the establishment of eumycetoma, or the mice andrabbits are not preferred hosts to strain TMMU3956. FulfillingKoch’s postulates through further experimental attempts isrequired to draw an etiologic conclusion.

In this study, a strong case has been made to demonstratethat strain TMMU3956 belongs to a novel species of Ma-durella, based on the macro- and micromorphological charac-teristic structures, temperature test, carbohydrate assimilationtest, and susceptibility testing. In addition, the multilocus DNAsequence comparisons and phylogenetic analysis give addi-tional evidence for concluding that strain TMMU3956 is a new

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species responsible for black-grain mycetoma and, as such,warrants further study. This strain has been deposited in theChina General Microbiological Culture Collection Center,Academia Sinica, Beijing, China, as CGMCC 3.12946 and inthe Centraalbureau voor Schimmelcultures, Utrecht, the Neth-erlands, as CBS 124574. The specific epithet Madurella pseudo-mycetomatis refers to the close relationship of the species toMadurella mycetomatis.

ACKNOWLEDGMENTS

We thank Mary Beth Neilly and Jianjun Chen (Medicine Section ofHematology/Oncology, University of Chicago, Chicago, IL) for assis-tance in reviewing the manuscript. We also thank G. S. de Hoog(Centraalbureau voor Schimmeccultures, Utrecht, the Netherlands)and Marie Desnos-Ollivier (Centre National de Reference Mycologieet Antifongiques, Unite de Mycologie Moleculaire, Institut Pasteur,Paris, France) for providing ITS sequences and microbiological datafor M. mycetomatis and M. grisea.

REFERENCES

1. Ahmed, A., D. Adelmann, A. Fahal, H. Verbrugh, A. van Belkum, and S. deHoog. 2002. Environmental occurrence of Madurella mycetomatis, the majoragent of human eumycetoma in Sudan. J. Clin. Microbiol. 40:1031–1036.

2. Ahmed, A. O., M. M. Mukhtar, M. Kools-Sijmons, A. H. Fahal, S. de Hoog,B. G. van den Ende, E. E. Zijlstra, H. Verbrugh, E. S. Abugroun, A. M.Elhassan, and A. van Belkum. 1999. Development of a species-specific PCR-restriction fragment length polymorphism analysis procedure for identifica-tion of Madurella mycetomatis. J. Clin. Microbiol. 37:3175–3178.

3. Ahmed, A. O., N. Desplaces, P. Leonard, F. Goldstein, S. De Hoog, H.Verbrugh, and A. van Belkum. 2003. Molecular detection and identificationof agents of eumycetoma: detailed report of two cases. J. Clin. Microbiol.41:5813–5816.

4. Ahmed, A. O., W. van Leeuwen, A. Fahal, W. van de Sande, H. Verbrugh, andA. van Belkum. 2004. Mycetoma caused by Madurella mycetomatis: a ne-glected infectious burden. Lancet Infect. Dis. 4:566–574.

5. Ahmed, A., W. van de Sande, H. Verbrugh, A. Fahal, and A. van Belkum.2003. Madurella mycetomatis strains from mycetoma lesions in Sudanesepatients are clonal. J. Clin. Microbiol. 41:4537–4541.

6. Ahmed, A. O., W. van Vianen, M. T. ten Kate, W. W. van de Sande, A. vanBelkum, A. H. Fahal, H. A. Verbrugh, and I. A. Bakker-Woudenberg. 2003.A murine model of Madurella mycetomatis eumycetoma. FEMS Immunol.Med. Microbiol. 37:29–36.

7. Ahmed, A. O., W. W. van de Sande, W. van Vianen, A. van Belkum, A. H. Fahal,H. A. Verbrugh, and I. A. Bakker-Woudenberg. 2004. In vitro susceptibilities ofMadurella mycetomatis to itraconazole and amphotericin B assessed by a mod-ified NCCLS method and a viability-based 2, 3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay. An-timicrob. Agents Chemother. 48:2742–2746.

8. Chen, Y. C., J. D. Eisner, M. M. Kattar, S. L. Rassoulian-Barrett, K. Lafe,S. L. Yarfitz, A. P. Limaye, and B. T. Cookson. 2000. Identification ofmedically important yeasts using PCR-based detection of DNA sequencepolymorphisms in the internal transcribed spacer 2 region of the rRNAgenes. J. Clin. Microbiol. 38:2302–2310.

9. Chen, Y. C., J. D. Eisner, M. M. Kattar, S. L. Rassoulian-Barrett, K. Lafe,U. Bui, A. P. Limaye, and B. T. Cookson. 2001. Polymorphic internal tran-

scribed spacer region 1 DNA sequences identify medically important yeasts.J. Clin. Microbiol. 39:4042–4051.

10. Conde, C., E. I. Melendro, M. Fresan, and L. Ortiz-Ortiz. 1982. Nocardiabrasiliensis: mycetoma induction and growth cycle. Infect. Immun. 38:1291–1295.

11. de Hoog, G. S., A. Buiting, C. S. Tan, A. B. Stroebel, C. Ketterings, E. J. deBoer, B. Naafs, R. Brimicombe, M. K. Nohlmans-Paulssen, G. T. Fabius, etal. 1993. Diagnostic problems with imported cases of mycetoma in TheNetherlands. Mycoses 36:81–87.

12. de Hoog, G. S., D. Adelmann, A. O. Ahmed, and A. van Belkum. 2004.Phylogeny and typification of Madurella mycetomatis, with a comparison ofother agents of eumycetoma. Mycoses 47:121–130.

13. Desnos-Ollivier, M., S. Bretagne, F. Dromer, O. Lortholary, and E. Dan-naoui. 2006. Molecular identification of black-grain mycetoma agents.J. Clin. Microbiol. 44:3517–3523.

14. Espinel-Ingroff, A., M. R. McGinnis, D. H. Pincus, P. R. Goldson, and T. M.Kerkering. 1989. Evaluation of the API 20C yeast identification system forthe differentiation of some dematiaceous fungi. J. Clin. Microbiol. 27:2565–2569.

15. Fahal, A. H. 2004. Mycetoma: a thorn in the flesh. Trans. R. Soc. Trop. Med.Hyg. 98:3–11.

16. Gumaa, S. A., E. S. Mahgoub, and M. A. el Sid. 1986. Mycetoma of the headand neck. Am. J. Trop. Med. Hyg. 35:594–600.

17. Lopes, M. M., G. Freitas, and P. Boiron. 2000. Potential utility of randomamplified polymorphic DNA (RAPD) and restriction endonuclease assay(REA) as typing systems for Madurella mycetomatis. Curr. Microbiol. 40:1–5.

18. Loulergue, P., A. Hot, E. Dannaoui, A. Dallot, S. Poiree, B. Dupont, and O.Lortholary. 2006. Successful treatment of black-grain mycetoma with vori-conazole. Am. J. Trop. Med. Hyg. 75:1106–1107.

19. Lynch, J. B. 1964. Mycetoma in the Sudan. Ann. R. Coll. Surg. Engl. 35:319–340.

20. National Committee for Clinical Laboratory Standards. 2002. Referencemethod for broth dilution antifungal susceptibility testing of filamentousfungi. Approved standard. Document M38-A. National Committee for Clin-ical Laboratory Standards, Wayne, PA.

21. Qin, Z. Y., S. X. Wu, R. L. Hopfer, G. X. Lv, C. J. Yao, and N. R. Guo. 2000.Glass beads-salting out procedure for extracting DNA from common patho-genic fungi. Chin. J. Dermatol. 33:330–332.

22. Rakeman, J. L., U. Bui, K. Lafe, Y. C. Chen, R. J. Honeycutt, and B. T.Cookson. 2005. Multilocus DNA sequence comparisons rapidly identifypathogenic molds. J. Clin. Microbiol. 43:3324–3333.

23. van de Sande, W. W., A. Luijendijk, A. O. Ahmed, I. A. Bakker-Woudenberg,and A. van Belkum. 2005. Testing of the in vitro susceptibilities of Madurellamycetomatis to six antifungal agents by using the Sensititre system in com-parison with a viability-based 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay and a mod-ified NCCLS method. Antimicrob. Agents Chemother. 49:1364–1368.

24. van de Sande, W. W., R. Gorkink, G. Simons, A. Ott, A. O. Ahmed, H.Verbrugh, and A. van Belkum. 2005. Genotyping of Madurella mycetomatisby selective amplification of restriction fragments (amplified fragment lengthpolymorphism) and subtype correlation with geographical origin and lesionsize. J. Clin. Microbiol. 43:4349–4356.

25. Vilela, R., O. M. Duarte, C. A. Rosa, J. G. Castro, S. Lyon, R. L. Motta, andA. C. Moura. 2004. A case of eumycetoma due to Madurella grisea in north-ern Brazil. Mycopathologia 158:415–418.

26. White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and directsequencing of fungal ribosomal RNA genes for phylogenetics, p. 315–322. InM. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols:a guide to methods and applications. Academic Press, San Diego, CA.

27. Zlotnik, H., and H. R. Buckley. 1980. Experimental production of actinomy-cetoma in BALB/c mice. Infect. Immun. 29:1141–1145.

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