Fungal myelitis caused by phialosimplex caninus in an immunosuppressed dog

Medical Mycology July 2012, 50, 509–512
Fungal myelitis caused by Phialosimplex caninus in an
immunosuppressed dog

PEDRO F. ARMSTRONG * , LYNNE SIGLER † , DEANNA A. SUTTON ‡ , AMY M. GROOTERS § & MARK HITT # * Southeast Veterinary Referral Center , Miami , FL , USA , University of Alberta Microfungus Collection and Herbarium , Edmonton , AB , Canada , Fungus Testing Laboratory , University of Texas Health Sciences Center , San Antonio , TX , § Veterinary Clinical Sciences ,Louisiana State University , Baton Rouge , LA , and # Atlantic Veterinary Internal Medicine , Annapolis , MD , USA A bone marrow infection caused by Phialosimplex caninus was diagnosed in a seven-year-old female spayed Cocker Spaniel that was receiving prednisone for autoimmune hemolytic anemia. Histopathologic examination of a bone marrow core biopsy revealed clusters of oval to round yeast-like cells of varying shape and size and occasional irregular hyphae. Culture of a bone marrow aspirate sample yielded a mould initially suggestive of Paecilomyces infl atus or Sagenomella species but later determined to be P. caninus . The dog was treated with itraconazole and amphotericin B, and pred-nisone was continued at the lowest dose needed to control the hemolytic anemia. The patient died after 18 months of treatment. This is the fi rst detailed clinical report of infection caused by P. caninus , a newly described fungus associated with disseminated disease in dogs. Keywords anemia , bone marrow biopsy , myelitis , Phialosimplex caninus , canine
forming solitary phialides [9 – 11]. The isolate in the present Introduction
case was one of six (fi ve canine and one human) that were Aspergillus , Paecilomyces , Penicillium and Geosmithia are included in the taxonomic study that led to the description members of the ascomycete family Trichocomaceae and of P. caninus and to reclassifi cation of Sagenomella chlamy- are among the most common moulds associated with dospora as P. chlamydosporus [8]. Because clinical details opportunistic infections in dogs [1 – 7]. Phialosimplex , a of the isolates were then unavailable, we report here the phylogenetically-related genus, was described recently and clinical history and unique aspects of histopathology of the contains two species, Phialosimplex caninus and Phialo- isolate that was selected as the type strain of P. caninus . simplex chlamydosporus , which are also associated with invasive canine infections [8]. All of these fungi are similar Med Mycol Downloaded from by University of Alberta on 07/16/12 in producing conidia in chains from phialides but in con- Case report
trast to the other genera, Phialosimplex species produce A seven-year-old, 14 kg female spayed Cocker Spaniel was conidia from solitary phialides that may be slightly swollen referred for evaluation of non-regenerative immune medi- centrally, and conidia are produced in both chains and clus- ated hemolytic anemia. The anemia had been diagnosed by ters [8]. Prior to the description of Phialosimplex , isolates the referring veterinarian three and a half months prior from cases of canine infection were reported incorrectly as based on a low hematocrit, spherocytosis and reticulocyto- being caused by Paecilomyces , Sagenomella or Monocil- sis. Antibody titers for Ehrlichia canis were negative, and lium species because each of these genera includes species a positive response to therapy with prednisone (1 mg/kg orally PO q12hrs) had initially been observed. At the time Received 16 July 2011 ; Received in fi nal form of referral, an acute decrease in the hematocrit and a retic- ulocyte count of less than 1% had been noted. Correspondence: Pedro F. Armstrong, Southeast Veterinary Referral Upon presentation to Atlantic Veterinary Internal Medi- Center, 6394 S. Dixie Hwy., Miami, FL 33143, USA. Tel.: ϩ 1 954 263 physical examination revealed hepatosplenomegaly 3658; Fax: ϩ 1 305 661 2434; E-mail: parmstrong@svrcfl 510 Armstrong et al.trong et al . and superfi cial cervical and popliteal lymphadenomegaly. A complete blood count revealed normocytic, normochromic anemia (hematocrit, 22%; reference interval: 36 – 60%), neu-trophilia (22,962/ μ l; reference interval: 2060 – 10,600/ μ l) with a left shift (band neutrophils 1335/ μ l; reference interval: 0 – 600/ μ l), and 3.6% corrected reticulocytes. Serum chemistry tests demonstrated an increased alkaline phosphatase (745 U/L; reference interval: 5–131 U/L) and results of urinalysis were unremarkable. Serum was negative for anti- Bartonella vinsonii antibody ( Ͻ 1:16), and PCR assays for Babesia and Ehrlichia genera performed on whole blood were negative. Abdominal sonography revealed hepatomegaly with normal echogenicity of the hepatic parenchyma, and mild intra-abdominal lymphadenopathy. The left limb of the pancreas was heterogeneous and surrounded by hyperechoic fat. Fine needle aspiration and cytologic evaluation of mesenteric and Fig. 1 Section from a bone marrow core biopsy stained with Gomori
popliteal lymph nodes revealed hyperplasia, with no evidence methenamine silver showing oval to round yeast-like fungal cells of varying size and rare fragments of irregular hyphae. Bar V 5 mm.
Histologic examination of a bone marrow core revealed severe chronic granulomatous myelitis with a marked mul- in-house) in culture tubes and a PFA slide culture incu- tifocal histiocytic infl ammatory infi ltrate, normocellular bated at 25 ° C for morphological examination. Additional erythroid and myeloid series, normal precursor cell matura- tests included assessment of growth of the isolate at tem- tion sequence, and adequate megakaryocytes. A Gomori peratures ranging from 25 – 40 ° C and on media containing methenamine silver stained preparation revealed prominent cycloheximide. Colonies, after 10 days of incubation, were clusters of oval to round yeast-like fungal organisms mea- somewhat granular, white to cream with a similar reverse, suring 3 – 12 μ m in diameter (Fig. 1). These cells produced slow growing with colony diameters less than 15 mm. one or more secondary cells suggestive of budding. Occa- Upon extended incubation a faint yellow diffusible pigment sional fragments of irregular hyphae or pseudohyphae was evident and the reverse became pale brownish. The were also noted. Serum antibody titers for Aspergillus , yellow diffusible pigment was more pronounced in tubes Blastomyces , Histoplasma , and Coccidioides , as well as than on plates. The isolate grew less well at 40 ° C than at serum Cryptococcus antigen titers, were all negative. Six 25, 30, or 35 ° C. No growth occurred on media containing days later, a bone marrow aspiration was performed to cycloheximide. Microscopically, globose conidia measur- obtain a sample for fungal culture. Cytologic evaluation of ing 2 – 3 μ m in diameter were borne in chains from some- this specimen again revealed the presence of similar fungal what infl ated mono- and polyphialides. The morphologic organisms and the remaining sample was submitted to the characteristics were originally suggestive of Paecilomyces Pythium Laboratory, Louisiana State University, for fungal infl atus or Sagenomella species. The isolate was sent for culture. A urine specimen collected at the same time was further evaluation to the University of Alberta Microfungus negative for bacterial and fungal growth. Therapy with itra- Collection and Herbarium (UAMH), Edmonton, AB, USA, conazole (Sporanox, Janssen Pharmaceutica, Piscataway, where it was accessioned as UAMH 10337 ( Med Mycol Downloaded from by University of Alberta on 07/16/12 NJ, USA) (5 mg/kg PO q24hr) was initiated, and prednisone 03-1073) and determined to be P. caninus following mor- administration (1 mg/kg orally PO q24hrs) was continued. phological and molecular comparison with related isolates At the Pythium Laboratory, a hyaline mould was iso- using sequencing of the internal transcribed spacer region lated on Sabouraud-dextrose agar after 48 h incubation at and the small subunit of the nuclear rRNA gene [8]. 37 ° C. Because a dimorphic pathogen was suspected based On day 16 after referral, treatment with amphotericin B on the yeast-like cells that had been observed in tissue, the lipid complex (Abelcet, The Liposome Company, Princeton, isolate was forwarded to the Fungus Testing Laboratory, NJ, USA) (1 mg/ml solution in 5% dextrose, 2.4 mg/kg, University of Texas Health Science Center, San Antonio, administered IV over 90 min three times weekly) was initi- Texas (UTHSC), for further identifi cation, and accessioned ated and itraconazole was discontinued. However after the into their culture collection as UTHSC 03-1073. Accu- sixth treatment, amphotericin had to be discontinued due to Probe DNA probes (Gen-Probe, Inc., San Diego, CA, development of azotemia. Urine cultures were negative for USA) for Histoplasma capsulatum and Blastomyces der- bacterial and fungal growth. Itraconazole was restarted at 5 matitidis carried out on the isolate were negative. The iso- mg/kg PO q24hr and prednisone was decreased to 0.5 mg/ late was subcultured onto potato fl akes agar (PFA, prepared kg/day. On days 40, 54 and 76 the hematocrit ranged from 2012 ISHAM, Medical Mycology, 50, 509–512
Myelitis caused by Phialosimplex caninus in a canine 511 23 – 27% (reference interval: 36 – 60%) and the creatinine and Shepherd dog has been caused by the related species Reevaluation on day 105 revealed persistent normocytic, The role of immunosuppressive therapy in the patho- normochromic anemia with minimal regenerative response genesis of infection in this case is not known as it was not (1.08% corrected reticulocytes). Bone marrow aspirate cytol- determined since infection was present prior to initiation ogy was normocellular with a normal erythroid maturation of prednisone therapy. However, given the breed predispo- sequence and no evidence of fungal organisms. Fungal cul- sition and initial hematologic characteristics, it seems most ture of the bone marrow sample yielded a mould that appeared likely that the anemia was originally the result of immune- morphologically similar to the initial isolate and therefore the mediated hemolytic anemia, and that infection with isolate was not forwarded for further evaluation. P. caninus then occurred subsequent to the administration Antifungal susceptibility testing performed at the Fungus of immunosuppressive therapy. Although P. caninus has Testing Laboratory using the published reference method not yet been isolated from an environmental source, infec- for testing fi lamentous fungi [NCCLS M38-A; 12], indi- tion is presumed to result from soil or airborne exposure. cated minimum inhibitory concentrations (MICs) in μ g/ml The unusual microscopic features, both in tissue and in of 0.03, 0.004, and 0.5 for itraconazole, terbinafi ne and culture, made the isolate in this case diffi cult to identify. The voriconazole, respectively. Although no defi ned break- presence of numerous oval to round yeast-like cells in tissue points are available for this organism, the isolate appeared and the isolation of a yellowish-white fi lamentous mould in to be susceptible to these agents (by human pharmacoki- culture initially led to concern that the organism was a dimor- netic standards) as MICs were within achievable serum phic pathogen but that diagnosis was ruled out by negative concentrations of the drugs using standard dosing regimens. reactions in the AccuProbe tests. The varying shape and size An MIC for fl uconazole at Ͼ 64 μ g/ml suggested no in vitro of the fungus and the production of one or more secondary cells suggests that P. caninus demonstrates a unique growth Periodic reevaluation by the referring veterinarian over form in tissue. Moreover, infections caused by P. caninus the next 11 months showed a stable hematocrit. Adding a appear to be characterized by presence of large numbers of second antifungal medication was discussed with the client pleomorphic forms. Similar observations were reported by but declined for fi nancial reasons. Itraconazole was con- Mackie et al . in a case in a Rottweiler of granulomatous tinued, and prednisone could not be tapered lower than lymphadenitis and splenitis that was tentatively attributed to 0.36 mg/kg orally every other day due to worsening of ane- Monocillium indicum but which was determined by Sigler mia. At reevaluation 440 days after referral, the dog contin- et al . as a probable case of P. caninus infection [8,11]. Simi- ued to have a normal activity level, appetite and water intake. larly, a fungus causing disseminated infection in a German Hematocrit was 18.4% (reference interval: 36 – 60%). Uri- nalysis showed pyuria and bacteriuria, and urine culture was species by Gersehenson et al . [13] based on the morphology positive for Escherichia coli , which was treated with amox- of yeast-like cells observed in hematoxylin and eosin stained icillin/clavulanic acid (Clavamox, Pfi zer Animal Health, sections, but the fungus was not grown in culture nor defi ni- New York, NY, USA) (15 mg/kg PO q12hr). Thoracic radio- tively identifi ed from tissue by immunohistochemistry or graphs were unremarkable, and systolic blood pressure was PCR. The description and illustrations of numerous budding normal. Abdominal sonography showed that the entire oval to round yeast-like cells measuring up to 10 μ m in diam- spleen was effaced with a coarse, complex, heteroechoic eter and the invasion of the fungus into lymph nodes, thoracic architecture. Therapy with itraconazole and prednisone was vertebrae, spleen and other organs is highly suggestive of Med Mycol Downloaded from by University of Alberta on 07/16/12 continued, but at 18 months after referral, the dog died of P. caninus infection as described in the present and previous unknown causes and necropsy was not performed. reports and emphasizes the invasive potential of the new agent of canine systemic disease [8,11]. The cultural features that distinguish P. caninus include Discussion
production of conidia from solitary phialides, development of This is the fi rst report to provide a detailed clinical history conidia in both chains and clusters, formation of yellowish- of a case of P. caninus infection. Among the fi ve cases of white colonies on potato fl ake agar and production of diffusible P. caninus disseminated canine infections known to date yellow pigments (Fig. 2). The phialides are narrow, cylindrical [8], this is the only one with evidence of bone marrow to slightly swollen centrally, tapering at the tip to an indistinct infection. Details on the clinical course of infection for collarette, and are typically monophialidic but sometimes the other cases are not available, but one dog was a Vizsla polyphalidic (i.e. having a second opening) (Fig. 3). Conidia and another was a German Shepherd. Three of the P. cani- are single-celled, smooth, hyaline, and produced in long chains nus isolates were obtained from lymph nodes and one or in clusters. The conidia differ in shape with those borne in from vertebrae. Similarly, discospondylitis in a German chains being subglobose and measuring 2.2 – 4 μm long by 2012 ISHAM, Medical Mycology, 50, 509–512
512 Armstrong et al.trong et al . late appeared susceptible in vitro to itraconazole, suggesting that initial treatment with this drug may be useful when infec-tion with P. caninus is suspected. However, despite the lack of fungal elements being seen in an aspirate collected during itra-conazole therapy, P. caninus was presumably still recovered in culture, although not submitted for identifi cation, suggesting suppression of the infection rather than eradication. Acknowledgements
L. Sigler thanks the Natural Sciences and Engineering Research Council of Canada for fi nancial support. Declaration of interest: The authors report no confl icts of
interest. The authors alone are responsible for the content
and writing of the paper.
Fig. 2 Phialosimplexcaninus (UAMH 10337) grown on potato dextrose
agar at 25°C for 14 days showing yellowish-white colonies and yellow
diffusible pigments.
1 Krockenberger MB, Swinney G, Martin P, Rothwell TR, Malik R. 1.8 – 3.7 μm wide and those in heads being obovoid and mea- Sequential opportunistic infections in two German Shepherd dogs. suring 2 – 4.5 μm long by 1.5 – 3.2 μm wide. Aust Vet J 2011; 89 : 9 – 14.
2 Schultz RM, Johnson EG, Wisner ER, et al . Clinicopathologic and In addition to providing new information about the clinical diagnostic imaging characteristics of systemic aspergillosis in 30 course of P. caninus canine infection, the case described here dogs . J Vet Intern Med 2008; 22 : 851 – 859.
is important for other reasons. First, although systemic fungal 3 Watt PR, Robins GM, Galloway AM, O ’ Boyle DA. Disseminated infection in an immunocompromised host usually carries a opportunistic fungal disease in dogs: 10 cases (1982 – 1990). J Am Vet poor prognosis, the dog in the present case survived 18 months Med Assoc 1995; 207 : 67 – 70.
4 Zanatta R, Miniscalco B, Guarro J, et al . A case of disseminated from the time of diagnosis despite the fact that prednisone mycosis in a German shepherd dog due to Penicillium purpurogenum . therapy had to be continued. It is also important to note that Med Mycol 2006; 44 : 93 – 97.
on the second bone marrow aspirate, fungal infection was 5 Booth MJ, van der Lugt JJ, van Heerden A, Pickard JA. Temporary detected by culture even though no organisms were noted cyto- remission of disseminated paecilomycosis in a German shepherd dog logically, suggesting that submission of bone marrow samples treated with ketoconazole. J S Afr Vet Assoc 2001; 72 : 99 – 104.
6 Foley JE, Norris CR, Jang SS. Paecilomycosis in dogs and horses and for fungal culture should be considered even if no organisms a review of the literature. J Vet Intern Med 2002; 16 : 238 – 243.
are visualized cytologically or histologically. Second, the iso- 7 Grant DC, Sutton DA, Sandberg CA, et al . Disseminated Geosmithia argillacea infection in a German Shepherd dog. Med Mycol 2009; 47 :
221 – 226.
8 Sigler, L., Sutton DA, Gibas CFC , et al . Phialosimplex , a new anamor- phic genus associated with infections in dogs and having phylogenetic
affi nity to the Trichocomaceae. Med Mycol 2010; 48 : 335 – 345.
9 Garcia ME, Caballero J, Toni P, et al . Disseminated mycoses in a dog by Paecilomyces sp. J Vet Med Physiol Pathol Clin Med 2000; 47 :
Med Mycol Downloaded from by University of Alberta on 07/16/12 10 Gen é J, Blanco J, Cano J, Garc í a ME, Guarro J. New fi lamentous fungus Sagenomella chlamydospora responsible for a disseminated
infection in a dog. J Clin Microbiol 2003; 41 : 1722 – 1725.
11 Mackie JT, Padhye AA, Sutherland RJ, Lamb WA, Davis S. Granu- lomatous lymphadenitis and splenitis associated with Monocillium
infection in a dog. J Vet Diagn Invest 2004; 16 : 248 – 250.
12 National Committee for Clinical and Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Fila-mentous Fungi, approved standard, document M38-A. Wayne, PA: National Committee for Clinical Laboratory Standards; 2002. Fig. 3 Microscopic morphology of Phialosimplex caninus (UAMH 10337)
Gershenson RT, Melidone R, Sutherland-Smith J, Rogers CL. showing production of conidia in chains and heads from narrow, simple, Abdominal aortic aneurysm associated with systemic fungal infection centrally swollen or cylindrical phialides. Bar V 5 mm.
in a German shepherd. J Am Anim Hosp Assoc 2011; 47 : 45 – 49.
This paper was fi rst published online on Early Online on 26 November 2011.
2012 ISHAM, Medical Mycology, 50, 509–512


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