Original Article Novel Penicillium Species Causing Disseminated

Medical Mycology, 2020, 0, 1–11 doi:10.1093/mmy/myaa016 Advance Access Publication Date: 0 2020 Original Article

10. Jiang, H., Chen, T.-J., Chi, Z., Hu, Z., Liu, G.-L., Sun, Y., Zhang, S.-H., and Chi, Z.-

M. (2019). Macromolecular pullulan produced by Aureobasidium melanogenum 13- Downloaded from https://academic.oup.com/mmy/advance-article-abstract/doi/10.1093/mmy/myaa016/5815442 by Briscoe Library user on 07 April 2020 2 isolated from the Taklimakan desert and its crucial roles in resistance to the stress Original Article treatments. International Journal of Biological Macromolecules 135: 429-436. Novel Penicillium species causing disseminated disease in a 11. Lu, Y., Wang, H., Wang, Z., Cong, Y., Zhang, P., Liu, G., Liu, C., Chi, Z.-M., and Labrador Retriever dog Chi, Z. (2019). Metabolic Rewiring Improves the Production of the Fungal Active Tatiana Rothacker 1, , Jared A Jaffey2, Erin R Rogers3, William H. Fales4, Targeting Molecule Fusarinine C. ACS Synth. Biol. 8: 1755-1765. ∗ Connie F. C. Gibas5, Nathan P.Wiederhold 5, Carmita Sanders5, James Mele5, 5 6 6 12. Zhang, Z., Lu, Y., Chi, Z., Liu, G.-L., Jiang, H., Hu, Z., and Chi, Z.-M. (2019). Hongxin Fan , Leah A. Cohn and Angela Royal Genome editing of different strains of Aureobasidium melanogenum using an efficient 1University of Missouri, A345 Clydesdale Hall, Columbia, Missouri, USA, 2Midwestern University, 19555 N 59th Ave, Cre/loxp site-specific recombination system. Fungal Biology 123: 723-731. Phoenix, Arizona, USA, 3University of Missouri, 2308 Houma Blvd 522, Metairie, Louisiana, USA, 4(Emeritus), Uni- versity of Missouri, 2328 Hamilton Drive, Ames, Iowa, USA, 5Fungus Testing Laboratory & Molecular Diagnostics 13. Chen, L., Chi, Z., Liu, G.-L., Xue, S.-J., Wang, Z.-P., Hu, Z., and Chi, Z.-M. (2019). Laboratory, Department of Pathology and Laboratory Medicine, University of Texas Health Science Center, San Improved pullulan production by a mutant of Aureobasidium melanogenum TN3-1 Antonio, Texas, USA and 6University of Missouri, A344 Clydesdale Hall, Columbia, Missouri, USA from a natural honey and capsule shell preparation. International Journal of ∗To whom correspondence should be addressed. Tatiana Rothacker, DVM, MS, University of Missouri, A345 Clydesdale Hall, Biological Macromolecules 141: 268-277. Columbia, Missouri 65211, USA. E-mail: [email protected]

Received 21 October 2019; Revised 7 February 2020; Accepted 2 March 2020; Editorial Decision 26 February 2020 14. Jia, S.-L., Ma, Y., Chi, Z., Liu, G.-L., Hu, Z., and Chi, Z.-M. (2019). Genome sequencing of a yeast-like fungal strain P6, a novel species of Aureobasidium spp.: Abstract Insights into its taxonomy, evolution and biotechnological potentials. Annals of This report describes the phenotypic characteristics of a novel Penicillium species, Penicillium labradorum, Microbiology 69:1475-1488. isolated from a 3-year-old male, castrated, Labrador retriever with disseminated fungal disease. The dog’s presenting clinical signs included lethargy, lymphadenopathy, tachypnea, moderate pitting edema, and non- weight bearing lameness associated with the right hind limb. Fine-needle aspirate biopsies from the sub- 15. Zhang, S., Jiang, H., Xue, S., Ge, N., Sun, Y., Chi, Z., Liu, G., and Chi, Z.-M. (2019). lumbar and prescapular lymph nodes were initially examined. The cytologic findings were consistent with Eﬃcient Conversion of Cane Molasses into Fructooligosaccharides by a Glucose pyogranulomatous inflammation with abundant extracellular and phagocytized fungal fragments andhy- Derepression Mutant of Aureobasidium melanogenum with High β‑Fructo- phae. Based on the morphology of the organisms and lack of endogenous pigment, hyalohyphomycosis furanosidase Activity. J. Agric. Food Chem. 67: 13665-13672. was considered most likely, with Fusarium, Penicillium, and Paecilomyces species being considerations. Fungal isolates were obtained via culture of samples from the lymph nodes, and molecular identification testing originally identified an undescribed Penicillium species belonging to the Penicillium section Exili- caulis. BLAST searches and phylogenetic analyses performed approximately 1 year and 9 months after the isolation date revealed an isolate within the Penicillium parvum clade in the Penicillium section Exilicaulis but phylogenetically distant from the other species in the section, thus representing a new species, Peni- cillium labradorum. Antifungal susceptibility testing was also performed on the isolate and low minimum inhibitory concentrations were observed with terbinafine, voriconazole, and posaconazole, while in vitro resistance was observed with fluconazole. The dog had been previously treated with fluconazole, itraconazole, amphotericin B lipid complex, voriconazole, and terbinafine. Approximately 587 days after the initial diagnosis, the dog was euthanized due to worsening of clinical signs and concerns for quality of life.

Key words: fungi, Penicillium, dog, lymphadenopathy.

Introduction azothiaprine, cyclosporine, etc.) have been suggested to be pre- 1-5 Penicillium is a ubiquitous fungus present in the general envi- disposing factors in dogs. German Shepherd dogs, particu- ronment with >300 known species.1 Most species of Penicillium larly young females, are susceptible to systemic fungal infec- are considered to be nonpathogenic (opportunistic) in humans tions, presumably due to a hereditary immunologic defect in this breed in which IgA immunoglobulins are decreased.1-5 and animals.2 Climatic conditions, genetically predisposed de- Other fects in immunity, and/or immunosuppressive therapies (steroids, genetically predisposed defects in immunity are generally not

© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. 1 All rights reserved. For permissions, please e-mail: [email protected] ______Reproduced from Medical Mycology (2020). Connie F. Cañete-Gibas: Participant of the 19th UM, 1991-1992.

350 351 2 Medical Mycology, 2020, Vol. 00, No. 00 recognized in most cases described in the veterinary literature.2,3 over 1 hour, three treatments per week for 4 weeks) and long- According to one review paper in which 157 cases of dis- term oral antifungal therapy with ketoconazole (1 g/day) and seminated mould infections in dogs were identified, 30 cases terbinafine (1 g/day).1 In the presented case, a previously unde- (19.1%) reported previous implementation of immunosuppres- scribed Penicillium species belonging to the Penicillium section sive therapy, 55 cases (35%) specifically stated that no pre- Exilicaulis was isolated. This report describes the phenotypic disposing factors were recorded, and 72 cases (45.9%) pro- characteristics of a new species, P. labradorum, as another agent Downloaded from https://academic.oup.com/mmy/advance-article-abstract/doi/10.1093/mmy/myaa016/5815442 by Briscoe Library user on 07 April 2020 vided no data on the immune status. In human medicine, most of disseminated fungal disease in a dog. cases of disseminated fungal disease have been attributed to a lack of suitable macrophage and/or neutrophil reaction in immunocompromised patients.5 More specific predisposing fac- Methods tors recognized in humans include acquired human immunod- Dog examined eficiency virus (HIV) infection, those on immunosuppressant therapy or cancer chemotherapy, neutropenic patients, or pa- A 3-year-old male castrated Labrador retriever was evaluated tients with poorly controlled diabetes mellitus. Some fungi re- by a primary care veterinarian for a 3-week history of lame- ported to cause disseminated disease in immunocompromised ness, lethargy, and lymphadenopathy. The dog recently moved humans include Aspergillus sp., Fusarium sp., Mucor sp., Cryp- to Kansas but had previously resided in Florida and California, tococcus sp., Penicillium sp., and Candida sp.6 Additionally, lo- USA. Pertinent physical examination findings at the time of ini- calized infections in otherwise healthy humans (usually follow- tial evaluation included mild tachypnea and moderately enlarged ing penetrating trauma) and infections of the nasal passages and firm prescapular, inguinal, and popliteal lymph nodes. Ul- and lower airways of dogs have also been described.7,8 In cases trasound guided fine-needle aspirate biopsies of an abdominal of disseminated disease, discospondylitis, osteomyelitis, and dif- lymph node were obtained and examined by the primary care fuse lymphadenopathy in dogs have been observed.1,3,9 Overall, veterinarian with cytologic recognition of suspected fungal hy- opportunistic filamentous fungal infections in dogs are infre- phae and pyogranulomatous inflammation. Treatment with flu- quently reported, with Aspergillus species being the most com- conazole (10 mg/kg PO q12hr) and doxycycline (5 mg/kg PO monly identified in cases of disseminated fungal disease.1,3,5 q12hr) were initiated at that time. Within 2 days of treatment, the The infrequent nature of Penicillium infections (with the ex- dog developed a depressed mentation and became increasingly ception of Talaromyces marneffei, formerly known as Penicil- more tachypneic and dyspneic. The dog was evaluated again lium marneffei) has been attributed to the inability of most by the primary care veterinarian and in addition to increased Penicillium species to grow at 37°C.9 Many of the previously respiratory abnormalities, moderate pitting edema and mild reported Penicillium species have recently been identified as weight bearing lameness associated with the right hindlimb were Talaromyces species infections. This includes: disseminated in- noted. There was also mild accumulation of edema along the fection clinically resembling multicentric malignant lymphoma ventral thorax and abdomen. Antifungal therapy was switched due to P. radicum (now identified as T. radicus),9 dissemi- from fluconazole to itraconazole and the patient was addition- nated mycosis in a German Shepherd due to P. purpurogenum ally prescribed carprofen (2.4 mg/kg PO q24hr) and tramadol (now identified as T.purpurogenus),3 intra-abdominal eumycotic (3.2 mg/kg PO q6–8hr) for pain management. The dog became mycetoma caused by P.duponti (now identified as Thermomyces anorectic and mildly ataxic in the following 2 days, thus prompt- dupontii),10 pulmonary infection with P. commune,11 and two ing referral to the University of Missouri Veterinary Health cases of osteomyelitis in which P. verruculosum (now identi- Center for further evaluation (day 1). Initial diagnostics during fied as T. verruculosus) and P.canis were isolated.1,2 A review this visit included a plasma biochemical profile and urinalysis of opportunistic fungal infections in 10 dogs also noted one case which were both unremarkable. A complete blood count (CBC) of nonspecified penicilliosis.4 The prognoses for these cases are revealed mild leukocytosis (19.17 K/μl; RI: 4.53–14.99 K/μl) poor, with cures unlikely despite aggressive anti-fungal therapy. characterized by a mild mature neutrophilia (15.14 K/μl; RI: Favorable outcomes with treatment, however, have been infre- 2.270–10.140 K/μl). Thoracic radiographs revealed mild perihi- quently reported and was initially seen in the present case fol- lar lymphadenopathy. Abdominal radiographs and ultrasonog- lowing multimodal medical therapy with amphotericin B lipid raphy showed sublumbar lymphadenopathy and multiple en- complex (Abelcet; 2 mg/kg, IV slow over 2 hours, 2–3 treatments larged intra-abdominal lymph nodes that measured up to 3.5 cm per week to total dose of 26 mg/kg) followed by long-term oral in diameter. Bony proliferations or other abnormalities associ- voriconazole (3.3 mg/kg PO q12hr) and terbinafine (29.2 mg/kg ated with the right hind limb were not observed on radiographs; PO q24hr). The dog reported to have infection caused by P. ca- therefore, it was concluded that the pitting edema present was nis also notably had a favorable outcome, with no clinical signs most likely due to impingement of the lymphatic system or oc- apparent 250 days after diagnosis following multimodal medical clusion of venous return by the enlarged sublumbar lymph nodes, therapy with amphotericin B lipid complex (Abelcet; 75 mg IV or less likely due to thrombus formation.

352 353 2 Medical Mycology, 2020, Vol. 00, No. 00 Rothacker et al. 3 recognized in most cases described in the veterinary literature.2,3 over 1 hour, three treatments per week for 4 weeks) and long- Sampling, cytology, immunological testing ited, Hampshire, UK), yeast extract sucrose (YES), and creatine According to one review paper in which 157 cases of dis- term oral antifungal therapy with ketoconazole (1 g/day) and Fine-needle aspirate biopsies of the sublumbar and prescapu- sucrose agar (CREA) (all prepared in-house). Colonial features 1 seminated mould infections in dogs were identified, 30 cases terbinafine (1 g/day). In the presented case, a previously unde- lar lymph nodes were submitted for evaluation and to confirm were examined after 7 days of incubation and the color recorded (19.1%) reported previous implementation of immunosuppres- scribed Penicillium species belonging to the Penicillium section fungal infection. All samples were collected using a 22-gauge nee- following the color descriptions in the Methuen Handbook of 13 sive therapy, 55 cases (35%) specifically stated that no pre- Exilicaulis was isolated. This report describes the phenotypic dle, and slide preparations were stained with a Wright-Giemsa Colour. Additional DNA sequencing and phylogenetic analy- Downloaded from https://academic.oup.com/mmy/advance-article-abstract/doi/10.1093/mmy/myaa016/5815442 by Briscoe Library user on 07 April 2020 Downloaded from https://academic.oup.com/mmy/advance-article-abstract/doi/10.1093/mmy/myaa016/5815442 by Briscoe Library user on 07 April 2020 disposing factors were recorded, and 72 cases (45.9%) pro- characteristics of a new species, P. labradorum, as another agent stain. Intraabdominal lymph node fine-needle aspirate biopsies sis are described below. Susceptibility testing was performed ac- 14 vided no data on the immune status. In human medicine, most of disseminated fungal disease in a dog. were repeated approximately 38 days later after finding multi- cording to the CLSI M38 reference standard. Scanning electron cases of disseminated fungal disease have been attributed to a ple enlarged intrabdominal lymph nodes on ultrasound exami- microscopy (SEM) was also used to obtain images of microscopic lack of suitable macrophage and/or neutrophil reaction in im- nation, with some being more enlarged than previously seen at structures. 5 munocompromised patients. More specific predisposing fac- Methods the initial referral. tors recognized in humans include acquired human immunod- Dog examined Immunological testing was pursued to screen the host’s im- Phenotypic characterization and molecular analysis eficiency virus (HIV) infection, those on immunosuppressant mune response to the pathogen. An immunoglobulin assay for A 3-year-old male castrated Labrador retriever was evaluated therapy or cancer chemotherapy, neutropenic patients, or pa- IgA, IgM, and IgG was performed through the Animal Health Mycelial mass from the isolates were harvested from PFA for by a primary care veterinarian for a 3-week history of lame- tients with poorly controlled diabetes mellitus. Some fungi re- Diagnostic Center of Cornell University. Other tests to eval- DNA extraction, and genomic DNA were extracted following ness, lethargy, and lymphadenopathy. The dog recently moved ported to cause disseminated disease in immunocompromised uate innate and adaptive immune functions included stimu- previously described protocols.15 The ITS rDNA region, partial to Kansas but had previously resided in Florida and California, humans include Aspergillus sp., Fusarium sp., Mucor sp., Cryp- lated leukocyte cytokine production capacity (Sigma-Aldrich, beta tubulin (BenA), calmodulin (CaM), and RNA polymerase 6 USA. Pertinent physical examination findings at the time of ini- tococcus sp., Penicillium sp., and Candida sp. Additionally, lo- St. Louis, MO, USA), leukocyte MHC-II surface expression II second largest subunit (RPB2) genes were amplified and se- tial evaluation included mild tachypnea and moderately enlarged calized infections in otherwise healthy humans (usually follow- (MHC-II antibody from Biorad, Hercules, CA, USA), lympho- quenced to compare with sequences of the same loci in previous and firm prescapular, inguinal, and popliteal lymph nodes. Ul- ing penetrating trauma) and infections of the nasal passages cyte blastogenesis (CD5 and ki67 antibodies from ThermoFisher, studies.16-18 Polymerase chain reaction (PCR) and sequenc- 7,8 trasound guided fine-needle aspirate biopsies of an abdominal and lower airways of dogs have also been described. In cases Waltham, MA, USA), and granulocyte/monocyte phagocytic ca- ing were carried out using primer pairs BMBC-R and NL4R lymph node were obtained and examined by the primary care of disseminated disease, discospondylitis, osteomyelitis, and dif- pacity (Phagotest®, Orpegen Pharma, Heidelberg, Germany), all (https://sites.duke.edu/vilgalyslab/rdna_primers_for_fungi/) for 1,3,9 veterinarian with cytologic recognition of suspected fungal hy- fuse lymphadenopathy in dogs have been observed. Overall, of which were compared to at least one healthy, age-matched ITS,19 BT2a and Bt2b for BenA,20 CF1 and CF4 for CaM,21 phae and pyogranulomatous inflammation. Treatment with flu- opportunistic filamentous fungal infections in dogs are infre- control dog evaluated concomitantly with the dog reported here. and 5F and 7CR for RPB2.22 The primer pair BMBC-R1 / NL42 conazole (10 mg/kg PO q12hr) and doxycycline (5 mg/kg PO quently reported, with Aspergillus species being the most com- Evaluation of granulocyte respiratory burst was performed us- was used since the amplification that is achieved results ina 1,3,5 q12hr) were initiated at that time. Within 2 days of treatment, the monly identified in cases of disseminated fungal disease. ing a commercially available test kit validated for use in canine complete sequence spanning both ITS1 and ITS2. This primer dog developed a depressed mentation and became increasingly The infrequent nature of Penicillium infections (with the ex- blood (Phagoburst®, Orpegen Pharma, Heidelberg, Germany)12 pair has been validated by the FTL for species identification of more tachypneic and dyspneic. The dog was evaluated again ception of Talaromyces marneffei, formerly known as Penicil- and compared to one healthy, age-matched, control dog. All test- clinical isolates received by this reference mycology laboratory. by the primary care veterinarian and in addition to increased lium marneffei) has been attributed to the inability of most ings were done after additional antifungal treatment while the Since this pair works very well, other primer pairs such as those 9 respiratory abnormalities, moderate pitting edema and mild 16,17 Penicillium species to grow at 37°C. Many of the previously patient was clinically well with no peripheral lymphadenopathy published by others were not used. The generated sequences weight bearing lameness associated with the right hindlimb were 23 reported Penicillium species have recently been identified as noted. were used to perform BLASTn searches in GenBank. BLASTn noted. There was also mild accumulation of edema along the Talaromyces species infections. This includes: disseminated in- search results were considered significant with an E-value of ventral thorax and abdomen. Antifungal therapy was switched fection clinically resembling multicentric malignant lymphoma 0.0, at 90–100% identity and from 90% query coverage. Phylo- from fluconazole to itraconazole and the patient was addition- due to P. radicum (now identified as T. radicus),9 dissemi- Fungal culture, morphology, and susceptibility testing genetic analyses were conducted independently with each locus ally prescribed carprofen (2.4 mg/kg PO q24hr) and tramadol nated mycosis in a German Shepherd due to P. purpurogenum Prior to the implementation of additional antifungal treatment, and combined to assess the relationship of isolates UTHSCSA 3 (3.2 mg/kg PO q6–8hr) for pain management. The dog became (now identified as T.purpurogenus), intra-abdominal eumycotic pre-scapular lymph node aspirates were placed in a BD-BACTEC DI19-20 and UTHSCSADI19-21 to members of the Penicillium anorectic and mildly ataxic in the following 2 days, thus prompt- mycetoma caused by P.duponti (now identified as Thermomyces PLUSTM antimicrobial removal device (Becton Dickinson Diag- section Exilicaulis. Sequences were aligned using MUSCLE 10 11 ing referral to the University of Missouri Veterinary Health dupontii), pulmonary infection with P. commune, and two nostics, Sparks, MD, USA) and a standard blood culture bot- as implemented in Sequencher ver 5.4.6 build 46289 (Gene Center for further evaluation (day 1). Initial diagnostics during cases of osteomyelitis in which P. verruculosum (now identi- tle and submitted for aerobic and anaerobic bacterial culture Codes Corp. Ann Arbor, MI, USA).24 Substitution models were 1,2 this visit included a plasma biochemical profile and urinalysis fied as T. verruculosus) and P.canis were isolated. A review and fungal culture through the University of Missouri Veterinary determined for each locus using the Finding Model program as which were both unremarkable. A complete blood count (CBC) of opportunistic fungal infections in 10 dogs also noted one case Medical Diagnostic Laboratory. The prescapular lymph node as- implemented in Molecular Evolutionary Genetics Analysis ver. 7 4 revealed mild leukocytosis (19.17 K/μl; RI: 4.53–14.99 K/μl) of nonspecified penicilliosis. The prognoses for these cases are pirates inoculated into the BD bottle produced a slow growing software (MEGA 7).25 Phylogenetic analyses using the maximum characterized by a mild mature neutrophilia (15.14 K/μl; RI: poor, with cures unlikely despite aggressive anti-fungal therapy. fungus and that was then subcultured to Sabouraud dextrose likelihood method based on the optimal evolutionary models 2.270–10.140 K/μl). Thoracic radiographs revealed mild perihi- Favorable outcomes with treatment, however, have been infre- agar (SDA) (RemelTM, Lenexa, KS, USA). Two fungal isolates for each locus and combined were conducted. The robustness of lar lymphadenopathy. Abdominal radiographs and ultrasonog- quently reported and was initially seen in the present case fol- were sent to the Fungus Testing Laboratory (FTL) at the Univer- the phylogenetic trees was evaluated by 1000 bootstrap resam- raphy showed sublumbar lymphadenopathy and multiple en- lowing multimodal medical therapy with amphotericin B lipid sity of Texas Health Science Center at San António, TX, USA for pling26 and Bayesian inference on the combined data set was larged intra-abdominal lymph nodes that measured up to 3.5 cm complex (Abelcet; 2 mg/kg, IV slow over 2 hours, 2–3 treatments species identification and susceptibility testing. The isolates were conducted in MrBayes v3.2.5 using the previously determined in diameter. Bony proliferations or other abnormalities associ- per week to total dose of 26 mg/kg) followed by long-term oral accessioned as UTHSCSA DI19-20 and UTHSCSA DI19-21. The optimal substitution model and the Markov chain Monte Carlo ated with the right hind limb were not observed on radiographs; voriconazole (3.3 mg/kg PO q12hr) and terbinafine (29.2 mg/kg specimens received at the FTL were subcultured onto potato algorithm.27 The analysis stopped when the average standard de- therefore, it was concluded that the pitting edema present was PO q24hr). The dog reported to have infection caused by P. ca- flakes agar (PFA) and incubated at 25°C and 37°C. Growthat viation of split frequencies reached 0.01. The sample frequency most likely due to impingement of the lymphatic system or oc- nis also notably had a favorable outcome, with no clinical signs 10°C, 25°C, 30°C, and 40°C was also assessed on Czapek yeast was set at 100 and the first 25% of trees were removed as clusion of venous return by the enlarged sublumbar lymph nodes, apparent 250 days after diagnosis following multimodal medical autolysate agar (CYA), malt extract agar (MEA, Oxoid Lim- burn-in. therapy with amphotericin B lipid complex (Abelcet; 75 mg IV or less likely due to thrombus formation.

352 353 4 Medical Mycology, 2020, Vol. 00, No. 00 Downloaded from https://academic.oup.com/mmy/advance-article-abstract/doi/10.1093/mmy/myaa016/5815442 by Briscoe Library user on 07 April 2020

Figure 1. A, B: Sublumbar lymph node aspirate biopsies from a dog with disseminated fungal infection caused by a novel Penicillium species, Penicillium labradorum sp. nov. Wright-Giemsa stain. (A) Macrophage containing many phagocytized fragments of fungal organisms. Many degenerate neutrophils are also present in the background. (B) Aggregates of extracellular fungal organisms with rare septate, parallel walled hyphal structures with bulbous ends (arrows). 100 objective. This Figure is reproduced in color in the online version of Medical Mycology. ×

Results Cytology, immunological testing, case outcome Cytologic preparations consisted primarily of aggregates of extracellular fungal organisms admixed with high numbers of nondegenerate and degenerate neutrophils, lower numbers of heavily vacuolated macrophages, and low numbers of lym- phocytes. Macrophages were often seen in aggregates and occasionally contained phagocytized fungal organisms (Fig. 1A). The fungal organisms were basophilic, round to oval, 4–5 mi- ∼ crons in diameter with irregular borders, and had a 1–2 micron thick, clear halo. Rare septate, parallel walled hyphal structures giving rise to bulbous ends were present (Fig. 1B). The cytologic diagnosis was pyogranulomatous inflammation with abundant intralesional fungal organisms, with hyalohyphomycosis Figure 2. Intraabdominal lymph node aspirates from a dog submitted 9 days considered most likely. after the initiation of antifungal therapy with itraconazole. Unlike the initial Repeat intraabdominal lymph node FNA biopsies consisted aspirate preparations, the extracellular fungal organisms are round to oval predominantly of macrophages containing phagocytized fungal and are now clear to homogeneously pale blue. Few macrophages, ruptured cells, and scattered erythrocytes are also present. Wright-Giemsa stain, 60 × organisms with morphologic features similar to those seen from objective. This Figure is reproduced in color in the online version of Medical the sublumbar lymph node aspirates submitted 1 month prior Mycology. ∼ (Fig. 2). Staining of the organisms, however, varied from the initial aspirates. They instead appeared clear to pale blue with rare Escherichia coli stimulated respiratory burst capacity compared sparse internal pink staining, which was presumed to be due to to a healthy age-matched control dog. All other tests evaluat- degenerative changes in the organisms as a consequence of the ing innate and adaptive immune functions were considered un- antifungal therapy. The patient was kept in hospital overnight remarkable when compared to at least one healthy, age-matched on IV fluid therapy and was discharged with unchanged antifun- control dog evaluated concomitantly. Control dogs were deter- gal therapy while the phenotyping and susceptibility panel was mined to be healthy based on a history reported by the owners pending. and the physical exam findings. An immunoglobulin assay for IgA, IgM, and IgG (performed Low minimum inhibitory concentrations, as measured by the through the Animal Health Diagnostic Center of Cornell Univer- CLSI M38 broth microdilution method, were observed with sity) revealed normal IgM and IgG levels and a slight decrease in terbinafine (0.03 μg/ml against both isolates), voriconazole (0.5 IgA (29 mg/dl; RI: 35–270 mg/dl). The slight decrease in IgA and 0.25 μg/ml, respectively), and posaconazole ( 0.03 and ≤ was not low enough to be considered clinically significant and 0.06 μg/ml, respectively), while in vitro resistance was ob- was not pursued further. Evaluation of granulocyte respiratory served with fluconazole (>64 μg/ml). An itraconazole blood con- burst, however, revealed that the dog had subjectively decreased centration (MVista®, Itraconazole bioassay, Indianapolis, IN,

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USA) was also evaluated in the dog to ensure recrudescence of species identification and susceptibility testing. At the FTL, mi- disease was not the result of inadequate therapeutic concentra- croscopic features were studied on PFA slide cultures incubated tions of itraconazole. While a specific therapeutic range for itra- for 3 and 7 days at 25°C and mounted in lactophenol cotton blue conazole in dogs is not known, blood concentrations >3.0 ug/ml (Fig. 3i,j). See Taxonomy below for culture description results. are recommended for systemic fungal infections when measured Top BLASTn searches with 0.0 E-values were 99% matched

Downloaded from https://academic.oup.com/mmy/advance-article-abstract/doi/10.1093/mmy/myaa016/5815442 by Briscoe Library user on 07 April 2020 by bioassay, and the dog in this report had an itraconazole with the following species, P. vinaceum CBS 389.49T, P. pimi- Downloaded from https://academic.oup.com/mmy/advance-article-abstract/doi/10.1093/mmy/myaa016/5815442 by Briscoe Library user on 07 April 2020 serum concentration of 10.6 ug/ml. Though the reported con- teouiense CBS 102479T, P. vinaceum CBS 389.48T, P. gut- centration represents a combination of both itraconazole and the tulosum NRRL 907T, P. parvum CBS 359 .48T, P. rubidurum active metabolite hydroxy-itraconazole (also found in the blood- CBS 609.73T for ITS, 91% matched with P. pimiteouiense CBS stream), a switch to amphotericin B lipid complex was rec- 102479T for CaM, 95% matched with P.vinaceum CBS 389.49T, ommended given these findings in conjunction with the dog’s P. parvum CBS 359 .48T and P. rubidurum CBS 609.73T for clinical progression of disease. BenA and 95% matched with P. vinaceum CBS 389.49T for

Figure 1. A, B: Sublumbar lymph node aspirate biopsies from a dog with disseminated fungal infection caused by a novel Penicillium species, Treatment with amphotericin B lipid complex began on day RPB2. Phylogenetic relationships were inferred using the Max- Penicillium labradorum sp. nov. Wright-Giemsa stain. (A) Macrophage containing many phagocytized fragments of fungal organisms. Many degenerate neu- 44, and the dog received 2 to 3 treatments per week for a cu- imum Likelihood based on the combined datasets of BenA, trophils are also present in the background. (B) Aggregates of extracellular fungal organisms with rare septate, parallel walled hyphal structures with bulbous mulative dosage of 26 mg/kg. During the course of amphotericin CaM, and RPB2 sequences and on individual data sets com- ends (arrows). 100 objective. This Figure is reproduced in color in the online version of Medical Mycology. × treatment, the dog’s appetite and attitude improved. The only ad- prising species in the P. parvum clade of the Penicillium sec- verse development was a focal thickening noted at the catheter tion Exilicaulis. The combined data sets had a total length of Results site after several treatments that eventually became ulcerated. 2154 characters (individual data sets: CaM 578; BenA 509; = = Punch biopsies of the lesion were obtained on day 51 and sub- RPB2 1067). Optimal substitution models based on the Cytology, immunological testing, case outcome = mitted for histopathology and for bacterial and fungal cultures. Akaike information criterion (AIC) were Kimura 2-parameter Cytologic preparations consisted primarily of aggregates of Histopathology revealed marked ulcerative dermatitis and ex- using a discreet Gamma distribution (K2P G) for BenA and + extracellular fungal organisms admixed with high numbers tensive vasculitis. No specific etiology was identified, though CaM, Kimura 2-parameter with invariant sites (K2P I) for + of nondegenerate and degenerate neutrophils, lower numbers a systemic process was suspected. Culture results yielded mul- RPB2 and Kimura 2-parameter using a discreet Gamma distribu- of heavily vacuolated macrophages, and low numbers of lym- tidrug resistant Staphylococcous pseudintermedius, and no fun- tion with invariant sites K2P G I for the combined datasets + + phocytes. Macrophages were often seen in aggregates and gal organisms were identified or cultured from the lesion. The of BenA, CaM and RPB2. occasionally contained phagocytized fungal organisms (Fig. 1A). dog was treated with chloramphenicol based on the culture and Results of the BLASTn searches and phylogenetic analyses The fungal organisms were basophilic, round to oval, 4–5 mi- susceptibility results, and skin lesions subsequently resolved en- showed that our isolates are within the P. parvum clade in the ∼ crons in diameter with irregular borders, and had a 1–2 micron tirely. Following successful delivery of a cumulative dosage of Penicillium section Exilicaulis but phylogenetically distant from thick, clear halo. Rare septate, parallel walled hyphal structures 26 mg/kg on day 79, amphotericin B lipid complex was discon- the other species in the section and represent a new species. The giving rise to bulbous ends were present (Fig. 1B). The cytologic tinued, and the dog was switched to voriconazole (3.3 mg/kg PO phylogram from the combined data sets showed isolates UTH- diagnosis was pyogranulomatous inflammation with abun- q12hr) and terbinafine (29.2 mg/kg PO q24hr). All clinical signs SCSA DI19-20 and UTHSCSA DI19-21 clustered together at dant intralesional fungal organisms, with hyalohyphomycosis Figure 2. Intraabdominal lymph node aspirates from a dog submitted 9 days of illness and all lympadenomegaly resolved completely. The dog 100% bootstrap support (BS) and 1.00 Bayesian posterior prob- considered most likely. after the initiation of antifungal therapy with itraconazole. Unlike the initial continued to do well for many months but moved away from ability value (BPP) and as a distinct clade and sister to P. parvum Repeat intraabdominal lymph node FNA biopsies consisted aspirate preparations, the extracellular fungal organisms are round to oval Missouri, and care was transferred to the University of Florida- at 92% (BS) and 1.00 BPP within the P. parvum clade (Fig. 4). predominantly of macrophages containing phagocytized fungal and are now clear to homogeneously pale blue. Few macrophages, ruptured cells, and scattered erythrocytes are also present. Wright-Giemsa stain, 60 Small Animal Hospital. After the dog had been in apparent re- Independent analyses of BenA, CaM, and RPB2 confirmed their × organisms with morphologic features similar to those seen from objective. This Figure is reproduced in color in the online version of Medical mission for well over a year, antifungal therapy was discontin- placement in the P. parvum clade as a distinct species clustered the sublumbar lymph node aspirates submitted 1 month prior Mycology. ued. Disease was confirmed to have recurred 535 days after the at 100% BS and closest to P. parvum but with low BS at <70% ∼ (Fig. 2). Staining of the organisms, however, varied from the ini- first amphotericin treatment. Although a plan was developed bootstrap for BenA, 80% for CaM and 84% with RPB2 (Fig. 5). tial aspirates. They instead appeared clear to pale blue with rare Escherichia coli stimulated respiratory burst capacity compared to restart treatment with amphotericin B, his clinical signs had sparse internal pink staining, which was presumed to be due to to a healthy age-matched control dog. All other tests evaluat- worsened quickly; he was hyporectic and developed a cough with Taxonomy degenerative changes in the organisms as a consequence of the a retch. Thoracic radiographs showed marked enlargement of ing innate and adaptive immune functions were considered un- Mycological assessment antifungal therapy. The patient was kept in hospital overnight remarkable when compared to at least one healthy, age-matched the mediastinal lymph nodes and a new nodule in the left lung on IV fluid therapy and was discharged with unchanged antifun- control dog evaluated concomitantly. Control dogs were deter- lobe. Due to the radiographic results and concerns for his quality Isolates UTHSCSA DI19-20 and UTHSCSADI19-21 had identi- gal therapy while the phenotyping and susceptibility panel was mined to be healthy based on a history reported by the owners of life, humane euthanasia was ultimately elected. cal sequences from all loci and were originally identified as an pending. and the physical exam findings. undescribed Penicillium species in section Exilicaulis based on An immunoglobulin assay for IgA, IgM, and IgG (performed Low minimum inhibitory concentrations, as measured by the DNA sequencing of the ITS rDNA region and a part of the beta through the Animal Health Diagnostic Center of Cornell Univer- CLSI M38 broth microdilution method, were observed with Macro- and micromorphology tubulin gene. sity) revealed normal IgM and IgG levels and a slight decrease in terbinafine (0.03 μg/ml against both isolates), voriconazole (0.5 The initial fungal culture performed at the University of Mis- IgA (29 mg/dl; RI: 35–270 mg/dl). The slight decrease in IgA and 0.25 μg/ml, respectively), and posaconazole ( 0.03 and souri yielded a slow growing fungus that formed white fungal Culture description ≤ was not low enough to be considered clinically significant and 0.06 μg/ml, respectively), while in vitro resistance was ob- colonies on SDA. The hyphae were sterile, septate, and devoid of Colony diameter: 7 days, in mm, 25°C: MEA 16–17; CYA was not pursued further. Evaluation of granulocyte respiratory served with fluconazole (>64 μg/ml). An itraconazole blood con- any identifying characteristics such as metulae, phialides, or coni- 10–11; YES 15–16; CREA 3–4. Colony characteristics: Mycelia burst, however, revealed that the dog had subjectively decreased centration (MVista®, Itraconazole bioassay, Indianapolis, IN, diophores, thus prompting submission of the isolates to FTL for white. Reddish brown (9E7) soluble pigments present on PFA

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Figure 3. Penicillium labradorum (UTHSCSA DI19-20). a–d. Colonies (obverse) at 30°C, 7 days, left to right: MEA, CYA, YES, and CREA. e–h. Colonies (reverse) 30°C, 7 days, left to right: MEA, CYA, YES, and CREA. i–j. Light micrographs of short conidiophores at 3 days (i) and 7 days (j). k–m. Scanning electron micrographs of short conidiophores and conidia. This Figure is reproduced in color in the online version of Medical Mycology.

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Figure 4. Maximum likelihood tree based on the combined dataset of BenA, CaM, and RPB2 sequences showing the isolates UTHSCSA DI19-20 and UTHSCSA DI19-21 placed within the Penicillium parvum clade of the Penicillium section Exilicaulis. Bootstrap values (left, 70%) and Bayesian posterior probabilities ≥ (right, 0.95 pp) are shown above the branches. Penicillium glabrum CBS 125543T was used as outgroup. CBS CBS culture collection, housed at the Westerdijk ≥ = Institute, Utrecht, The Netherlands, NRRL Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, US Department = of Agriculture, 1815 North University Street, Peoria, IL 61604, USA, DAOMC Canadian Collection of Fungal Cultures, IMI International Mycological institute = = (CABI Centre for Agriculture and Bioscience International, UK). T type species. This Figure is reproduced in color in the online version of Medical Mycology. = = but absent on MEA, CYA, and YES. On PFA, colonies were gray (3D2–3D3) on obverse, olivaceous (3D3–3D4) on reverse flat and floccose, producing sparse to moderately dense sporu- (Fig. 3c,g) and showed a similar obverse pattern as on CYA. lation, with conidia dull greyish green (30E4 obverse) and en Colonies on CREA were restricted attaining only 3–4 mm in di- masse and reddish brown reverse (8E8). On CYA, colonies grew ameter after 7 days, flat and greenish gray obverse (30C3) and slowly attaining a diameter of 10–11 mm, erumpent, floccose, grayish brown (6D3) reverse, acid and base production were not folded, wrinkled, and crateriform, appearing dull cream with an observed (Fig. 3d,h). Figure 3. Penicillium labradorum (UTHSCSA DI19-20). a–d. Colonies (obverse) at 30°C, 7 days, left to right: MEA, CYA, YES, and CREA. e–h. Colonies (reverse) olive (3D3) perimeter (Fig. 3b,f). Colonies on MEA were low, Micromorphology: Conidiophores were monoverticillate 30°C, 7 days, left to right: MEA, CYA, YES, and CREA. i–j. Light micrographs of short conidiophores at 3 days (i) and 7 days (j). k–m. Scanning electron micrographs with radial folds, centrally raised, displaying greenish gray to with smooth short stipes (7.5–15 um in length); metulae ab- of short conidiophores and conidia. This Figure is reproduced in color in the online version of Medical Mycology. olive (3E3) at the periphery and pale orange (6A3) towards the sent, phialides smooth, ampulliform, 2–4 per stipe (3.2–5 um center (Fig. 3a,e), yellowish brown reverse (5E4) with moder- in length). Conidia were finely roughened, globose to subglo- ate to dense sporulation. Growth on YES was yellowish olive bose (1.9–2.3um 1.9–2.3um). No sclerotia or ascomata were ×

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Figure 5. Maximum likelihood trees based on the individual data sets of BenA, CaM, and RPB2 sequences showing the relationship of the isolates UTHSCSA DI19-20 and UTHSCSA DI19-21 with species within the Penicillium parvum clade of the Penicillium section Exilicaulis. The numbers to the left of the species names are GenBank accession numbers. Bayesian posterior probabilities (Left, 0.95 pp) and Bootstrap values (Right, 70%) are shown above the branches. ≥ ≥ The trees are rooted with Penicillium glabrum CBS 125543T. This Figure is reproduced in color in the online version of Medical Mycology.

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observed on all media after >6 months incubation. Growth was line hyphae lacking pigment within the walls are a common char- good on YES at 37°C but slower on PFA, CYA, and MEA and acteristic, hence categorizing it as one of many organisms which variable at 40°C. Short conidiophores and conidia were also vi- cause “hyalohyphomycosis.” This term is used as a counterpart sualized with SEM (Fig. 3k–m). to the term “phaeohyphomycosis,” in which fungi appear in tis- Based on phylogenetic results and their distinct phenotypic sues as septate hyphae containing an endogenous pigment. In the

Downloaded from https://academic.oup.com/mmy/advance-article-abstract/doi/10.1093/mmy/myaa016/5815442 by Briscoe Library user on 07 April 2020 characteristics, we describe these two isolates as a new species presented case, the preservation of the fungal hyphal morphol- Downloaded from https://academic.oup.com/mmy/advance-article-abstract/doi/10.1093/mmy/myaa016/5815442 by Briscoe Library user on 07 April 2020 named Penicillium labradorum sp. nov. ogy was poor on cytology, with only fragments and rare septate, branching, nonpigmented fungal hyphae seen (possibly the result of prior treatment with itraconazole). On the basis of the fun- Penicillium labradorum gal characteristics that were evident, likely differential diagnoses Gibas, Wiederhold, Sanders, Rothacker, Rogers et Fales sp. nov. included infection with Penicillium, Fusarium, or Paecilomyces Mycobank: MB831086 species. Aspergillus was ruled out based on the lack of charac- In: Penicillium subgenus Aspergilloides section Exilicaulis. teristic 45° angle branching associated with that genus.2 Penicillium species in the section Exilicaulis are character- Etymology ized by having mono and biverticillate conidiophores and non- vesiculated stipes.28 Some species in this section have been im- Named after a Labrador retriever dog where it was isolated from. plicated in several conditions such as allergies from extrolites (e.g., citreoviridin, a mycotoxin extrolite known to cause yel- Type and holotype low rice disease or cardiac beri-beri in humans [P. citreoni- Missouri, United States of America, from prescapular lymph grum]),29 osteomyelitis in dogs (P. canis),1 and para-vertebral node aspirates from a Labrador retriever dog with disseminated infection and mycetoma (fungus ball) in humans (P. decum- infection. Collected by D.V.M. Erin Rogers (June 11, 2017) and bens).30,31 Penicillium parvum is the closest relative of P.labrado- isolated by PhD William Fales (July 17, 2017). Holotype CBS rum. Phenotypically, P. labradorum is similar to P. parvum by = H-24321. Culture ex-type UTHSCSA DI19-20 CBS 145775, its slow growth, production of a reddish-brown diffusing pig- = preserved in a metabolically inactive state at the CBS cul- ment, and short conidiophores. However, P. labradorum dif- ture collection, Westerdijk Fungal Biodiversity Institute, The fers by its production of finely roughened conidia whereas Netherlands. ITS barcode: MK881918; Alternative markers: P. parvum produces smooth conidia.32 Although Penicillium = BenA MK887898, CaM MK887899, RPB2 MK887900. parvum has a sexual state, the sexual state of P. labradorum was = = = not observed in our isolates after >6 months of incubation in all media used; however, mating studies were not performed. Other strains In addition, P. labradorum is phylogenetically distant from P. UTHSCSA DI19-21 CBS 145776. ITS barcode: MK882920 = = parvum having 24 base pair differences in partial CaM sequences Alternative markers: BenA MK887901, CaM MK887902, = = and 45 base pair differences in both partial BenA and RPB2 RPB2 MK887903. = sequences. The two strains examined had identical ITS, BenA, CaM and Identification of fungal isolates is usually achieved via exam- RPB2 sequences. ination of fruiting structures formed in culture, although evaluation of fungal culture by morphology combined with PCR Distribution and ecology amplification and sequencing are considered as the criterion standard for fungal identification.2,33 In the present case, molecular Isolated in the United States and distribution in other parts of and morphological methods were used to identify the organisms. the world is unknown. In the beginning, the primary cultures only produced sterile hyphae with no microscopic features to assist with identification. Discussion The lack of identifying features may be attributed to the previous In this report, we describe a new species of Penicillium, P. treatment with antifungals prior to the samples being submitted labradorum sp. nov., within the section Exilicaulis. As described for culture. in the case, the morphology of the organism on cytology was Additionally, the possibility of culture growth due to con- most consistent with an organism causing hyalohyphomycosis, tamination was considered as Penicillium is a common labo- with fungal culture and isolation yielding a previously unidenti- ratory contaminant. For the dog in this report, however, this

Figure 5. Maximum likelihood trees based on the individual data sets of BenA, CaM, and RPB2 sequences showing the relationship of the isolates UTHSCSA fied Penicillium sp. Morphologically, Penicillium is described as was considered less likely since the morphological features of DI19-20 and UTHSCSA DI19-21 with species within the Penicillium parvum clade of the Penicillium section Exilicaulis. The numbers to the left of the species having septate hyphae (2–5 μm in width) giving rise to branched cultured isolates were similar to those observed on cytology names are GenBank accession numbers. Bayesian posterior probabilities (Left, 0.95 pp) and Bootstrap values (Right, 70%) are shown above the branches. ≥ ≥ or unbranched conidiophores with secondary branches, which (sterile hyalinized hyphae lacking pigment), and the findings of The trees are rooted with Penicillium glabrum CBS 125543T. This Figure is reproduced in color in the online version of Medical Mycology. give it a brush-like appearance in culture.1 In tissues, septate hya- these organisms were repeated on both the initial and follow-up

358 359 10 Medical Mycology, 2020, Vol. 00, No. 00 cytologic examination from the sublumbar and intraabdominal azoles (e.g., P. citrinum, P. oxalicum, P. rubens) and some re- lymph nodes. When the cultures were submitted to the FTL for ports showing resistance to voriconazole.33,39 This case is unique identification and were subsequently subcultured onto various given the cytologic findings, the novel Penicillium sp. isolated, culture media for identification workup, the isolates grew nor- and the dog’s diminished granulocyte respiratory burst function- mally as a typical Penicillium species (i.e., producing structures ality compared to a normal dog. Previous cases investigating res- salient to Penicillium species). piratory burst functionality as a causative factor of S. pseudinter- Downloaded from https://academic.oup.com/mmy/advance-article-abstract/doi/10.1093/mmy/myaa016/5815442 by Briscoe Library user on 07 April 2020 Multiple immunologic processes are required to successfully medius infections and disseminated fungal disease in dogs were clear fungal diseases including opsonization, phagocytosis, and not found in the literature. Although it is suspected that the di- destruction of fungal organisms utilizing oxidative damage by minished granulocyte respiratory burst capacity predisposed this a respiratory burst.6,34 Therefore, substantial disruptions in im- dog to disseminated fungal disease and possibly secondary infec- mune function at any of these stages could be significant enough tion with S. pseudintermedius, additional testing would be nec- to prevent disease clearance, with subsequent dissemination of essary to more definitively determine the role of an immune de- fungal organisms being more likely. The immune function as- ficiency of this nature as the results were compared to onlyone sessment in this patient suggested that there was suppression of healthy, age-matched control dog. granulocyte respiratory burst capacity. It is suspected that the diminished respiratory burst in this dog increased susceptibility to fungal disease. It is uncertain if the systemic fungal disease Acknowledgments further suppressed the dog’s immune system, or if the same res- The authors thank Dr.Amy DeClue and Dr.Carol Reinero at the University piratory burst defect played a permissive role in the secondary of Missouri-Veterinary Health Center for providing the assays, lab time, and materials for the immunological testing performed, Hans Rindt for bacterial infection with S. pseudintermedius. Iatrogenic infection performing most of the immunological testing mentioned in this paper, and with this bacteria also could not be entirely ruled out. Aside from Barbara Hunter for performing the scanning electron microscopy. prior treatment with steroids,11 other predisposing defects in immunity have not been recognized in previous reports of Penicil- lium sp. infections in dogs, making this an interesting finding. Declaration of interest It is known that some fungal pathogens can exert an ability to The authors report no conflicts of interest. The authors alone are respon- mask themselves from the immune system, or dampen immune sible for the content and the writing of the paper. function to enhance survivability. One such fungal organism is Candida albicans, which actively suppresses the production of reactive oxygen species involved in the respiratory burst.35 Vari- References ation of virulence is also known to occur within the genus and 1. Langlois DK, Sutton DA, Swenson SL et al. Clinical, morphological, and molecular characterization of Penicillium canis sp. nov., isolated from a dog with os- species of fungal organisms. For example, Aspergillus fumigatus teomyelitis. J Clin Microbiol. 2014; 52: 2447–2453. has been demonstrated to be less pathogenic than A. nidulans in 2. Miyakawa K, Swenson CL, Mendoza L, Boyle MH, Steficek BA. 2011. Pathology human patients with chronic granulomatous disease (a disease in practice: penicilliosis. J Am Vet Med Assoc. 238: 51–53. process with features similar to the presented case in which the 3. Zanatta R, Miniscalco B, Guarro J et al. A case of disseminated mycosis in a Ger- man Shepherd dog due to Penicillium purpurogenum. Medical Mycology. 2006; 36 functionality of the respiratory burst pathway is disrupted). In 44: 93–97. this patient, a novel species of Penicillium resulted in dissemi- 4. Watt PR, Robins GM, Galloway AM, O’Boyle DA. Disseminated opportunistic nated disease. This leaves the possibility of a more pathogenic fungal disease in dogs: 10 cases (1982–1990). J Am Vet Med Assoc. 1995; 207: 67–70. species of Penicillium in general, or perhaps a species of Penicil- 5. Elad D. Disseminated canine mold infections. Vet J. 2019; 243: 82–90. lium that is able to exploit this patient’s particular immune com- 6. Lionakis MS, Iliev ID, Hohl TM. Immunity against fungi. JCI Insight. 2017; 2: promise as an alternative scenario to opportunistic infection. 1–17. 7. Nucci M, Anaissie EJ. Chapter 13: Hyalohyphomycosis. In: Anaissie EJ, McGin- It is uncertain if the long-term favorable response to treatment nis MG Pfaller MA, eds. Clinical Mycology. 2nd ed. Churchill Livingstone: Else- in this case was a result of the species of fungal organism in- vier Inc. 2009, 309–327. volved or the aggressive treatment protocol. The in vitro suscep- 8. Harvey CE, O’Brien JA, Felsburg PJ et al. Nasal penicilliosis in six dogs. J Am tibilities measured against the two P. labradorum isolates in this Vet Med Assoc. 1981; 178: 1084–1087. 9. De Vos JP, Van Garderen E, Hensen H et al. Disseminated penicillium radicum in- case are similar to what has previously been reported against P. fection in a dog, clinically resembling multicentric malignant lymphoma. Vlaams canis, with potent activity observed with terbinafine, posacona- Diergeneeskundig Tijdschrift. 2009; 78: 183–187. zole, and voriconazole, and in vitro resistance to fluconazole.1 10. Janovec J, Brockman DJ, Priestnall SL, Kulendra NJ. Successful treatment of intra-abdominal eumycotic mycetoma caused by Penicillium duponti in a dog. Terbinafine and posaconazole have also been reported by oth- J Small Anim Pract. 2016; 57: 159–162. ers to have good in vitro activity against Penicillium species.37,38 11. Kano R, Ito K, Imagi M et al. Isolation of Penicillium commune from a pulmonary In contrast, others have reported that the activities of voricona- infection in a dog. Vet Rec. 2006; 159: 779–780. 12. LeBlanc CJ, LeBlanc AK, Jones MM, Bartges JW, Kania SA. Evaluation of pe- zole and posaconazole may be variable against Pencillium with ripheral blood neutrophil function in tumor-bearing dogs. 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360 361 10 Medical Mycology, 2020, Vol. 00, No. 00 Rothacker et al. 11 cytologic examination from the sublumbar and intraabdominal azoles (e.g., P. citrinum, P. oxalicum, P. rubens) and some re- 13. Kornerup A, Wanscher JH. Methuen Handbook of Colour, 3rd ed. London: Eyre 26. Felsenstein J. Confidence limits on Phylogenies: an approach using the bootstrap. lymph nodes. When the cultures were submitted to the FTL for ports showing resistance to voriconazole.33,39 This case is unique Methuen Ltd, 1978. Evolution 1985; 39: 783–791. 14. CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of 27. Geyer C. Introduction to markov chain monte carlo. In: Brooks S, Gelman A, identification and were subsequently subcultured onto various given the cytologic findings, the novel Penicillium sp. isolated, Filamentous Fungi. Wayne,PA, USA: Clinical and Laboratory Standards Institute; Jones G Meng XL eds. Handbook of Markov Chain Monte Carlo. 1st ed. Chapel culture media for identification workup, the isolates grew nor- and the dog’s diminished granulocyte respiratory burst function- 2017. Hill, NC, USA: Chapman and Hall/CRC, 2011. mally as a typical Penicillium species (i.e., producing structures ality compared to a normal dog. Previous cases investigating res- 15. Ahmed B, Gupta N, Gibas CFC et al. Rasamsonia sp: an emerging infec- 28. Houbraken J, Samson RA. Phylogeny of Penicillium and the segregation of tri- tion amongst chronic granulomatous disease patients. A case of disseminated chocomaceae into three families. Stud Mycol. 2011; 70: 1–51. salient to Penicillium species). piratory burst functionality as a causative factor of S. pseudinter- Downloaded from https://academic.oup.com/mmy/advance-article-abstract/doi/10.1093/mmy/myaa016/5815442 by Briscoe Library user on 07 April 2020 Downloaded from https://academic.oup.com/mmy/advance-article-abstract/doi/10.1093/mmy/myaa016/5815442 by Briscoe Library user on 07 April 2020 infection by a putatively novel Rasamsonia argillacea species complex involving 29. Kushiro M. Historical review of researches on yellow rice and mycotoxigenic Multiple immunologic processes are required to successfully medius infections and disseminated fungal disease in dogs were the heart. Med Mycol Case Rep. 2019; 24: 54–57. fungi adherent to rice in Japan. JSM Mycotoxins 2015; 65: 19–23. clear fungal diseases including opsonization, phagocytosis, and not found in the literature. Although it is suspected that the di- 16. Visagie CM, Seifert KA, Houbraken J et al. A phylogenetic revision of Penicillium 30. Lyratzopoulos G, Ellis M, Nerringer R, Denning DW. Invasive infection due to destruction of fungal organisms utilizing oxidative damage by minished granulocyte respiratory burst capacity predisposed this sect. Exilicaulis, including nine new species from fynbos in South Africa. IMA Penicillium species other than P. marneffei. J Infect. 2002; 45: 184–195. Fungus. 2016; 7: 75–117. 31. Yoshida K, Hiraoka T, Ando M et al. Penicillium decumbens. Chest. 1992; 101: 6,34 a respiratory burst. Therefore, substantial disruptions in im- dog to disseminated fungal disease and possibly secondary infec- 17. Houbraken J, Seifert KA, Samson RA. Penicillium hermansii, a new species caus- 1152–1153. mune function at any of these stages could be significant enough tion with S. pseudintermedius, additional testing would be nec- ing smoky mould in white button mushroom production. Mycological Progress. 32. Raper K, Fennell D. New species of Penicillium. Mycologia. 1948; 40: 507–546. to prevent disease clearance, with subsequent dissemination of essary to more definitively determine the role of an immune de- 2019; 18: 229–236. 33. Guevara-Suarez M, Sutton DA, Cano-Lira JF et al. Identification and antifungal 18. Visagie CM., Renaud JB, Burgess KMN et al. Fifteen new species of Penicillium. susceptibility of Penicillium-like fungi from clinical samples in the United States. fungal organisms being more likely. The immune function as- ficiency of this nature as the results were compared to onlyone Persoonia. 2016; 36: 247–280. J Clin Microbiol. 2016; 54: 2155–2161. sessment in this patient suggested that there was suppression of healthy, age-matched control dog. 19. Lane DJ, Pace B, Olsen GJ et al. Rapid determination of 16S ribosomal RNA 34. Blanco JL, Garcia ME. Immune response to fungal infections. Vet Immunol Im- granulocyte respiratory burst capacity. It is suspected that the sequences for phylogenetic analyses. Proc. Natl. Acad. 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J Innate Immun. 2018; 10: piratory burst defect played a permissive role in the secondary of Missouri-Veterinary Health Center for providing the assays, lab time, species isolated from a coffee plant and its phylogenetic relationship to P. fellu- 145–160. and materials for the immunological testing performed, Hans Rindt for tanum, P. thiersii and P. Brocae based on parsimony analysis of multilocus DNA 37. Diekema DJ, Messer SA, Hollis RJ et al. Activities of caspofungin, itraconazole, bacterial infection with S. pseudintermedius. Iatrogenic infection performing most of the immunological testing mentioned in this paper, and sequences. Mycologia. 2005; 97: 659–666. posaconazole, ravuconazole, voriconazole, and amphotericin B against 448 re- with this bacteria also could not be entirely ruled out. Aside from Barbara Hunter for performing the scanning electron microscopy. 22. Liu YJ, Whelen S, Hall BD. Phylogenetic relationships among ascomycetes: evi- cent clinical isolates of filamentous fungi. J Clin Microbiol. 2003; 41: 3623–3626. prior treatment with steroids,11 other predisposing defects in im- dence from an RNA polymerase II subunit. Mol Biol Evol. 1999; 16: 1799–1808. 38. Cuenca-Estrella M, Gomez-Lopez A, Mellado E et al. Head-to-head comparison 23. Altschul SFP, Gish W, Miller W et al. Basic local alignment search tool. J Mol of the activities of currently available antifungal agents against 3,378 Spanish munity have not been recognized in previous reports of Penicil- Biol. 1990; 215: 403–410. clinical isolates of yeasts and filamentous fungi. Antimicrob Agents Chemother. lium sp. infections in dogs, making this an interesting finding. Declaration of interest 24. Edgar RC. MUSCLE: A multiple sequence alignment method with reduced time 2006; 50: 917–921. It is known that some fungal pathogens can exert an ability to The authors report no conflicts of interest. The authors alone are respon- and space complexity. BMC Bioinformatics. 2004; 5: 113. 39. Chowdhary A, Kathuria S, Agarwal K et al. Voriconazole-resistant Penicillium 25. Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics anal- oxalicum: an emerging pathogen in immunocompromised hosts. Open Forum mask themselves from the immune system, or dampen immune sible for the content and the writing of the paper. ysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33: 1870–1874. Infect Dis. 2014; 1: 1–7. function to enhance survivability. One such fungal organism is Candida albicans, which actively suppresses the production of reactive oxygen species involved in the respiratory burst.35 Vari- References ation of virulence is also known to occur within the genus and 1. Langlois DK, Sutton DA, Swenson SL et al. Clinical, morphological, and molecular characterization of Penicillium canis sp. nov., isolated from a dog with os- species of fungal organisms. 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De Vos JP, Van Garderen E, Hensen H et al. Disseminated penicillium radicum in- case are similar to what has previously been reported against P. fection in a dog, clinically resembling multicentric malignant lymphoma. Vlaams canis, with potent activity observed with terbinafine, posacona- Diergeneeskundig Tijdschrift. 2009; 78: 183–187. zole, and voriconazole, and in vitro resistance to fluconazole.1 10. Janovec J, Brockman DJ, Priestnall SL, Kulendra NJ. Successful treatment of intra-abdominal eumycotic mycetoma caused by Penicillium duponti in a dog. Terbinafine and posaconazole have also been reported by oth- J Small Anim Pract. 2016; 57: 159–162. ers to have good in vitro activity against Penicillium species.37,38 11. Kano R, Ito K, Imagi M et al. Isolation of Penicillium commune from a pulmonary In contrast, others have reported that the activities of voricona- infection in a dog. Vet Rec. 2006; 159: 779–780. 12. LeBlanc CJ, LeBlanc AK, Jones MM, Bartges JW, Kania SA. 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