Granulomatous polyarthritis caused by Talaromyces georgiensis in a dog

Abstract

A 6-y-old, 3.5-kg, spayed female Toy Poodle was presented with left forelimb lameness of 2-d duration. Two months before the initial presentation, radiography showed osteolysis of the medial epicondyle of the left humerus, and the left forelimb was amputated. Grossly, the articular villi of the elbow joint were markedly thickened, and the articular cartilage surfaces of the distal humerus and proximal radius had partial erosion. Histologically, granulomatous arthritis and osteomyelitis characterized by the presence of abundant macrophages containing numerous fungi were observed. ITS and β-tubulin sequences amplified from the isolate from the specimen were 100% and 99% identical to type strain UTHSC D16-145^T of Talaromyces georgiensis, respectively. Canine osteoarthritis caused by T. georgiensis has not been reported previously, to our knowledge.

Genus Talaromyces (family Trichocomaceae ¹² ), known previously as an asexual genus of Penicillium, has been isolated from soil and insects worldwide. ³ Some Talaromyces spp. are pathogenic, and have been reported to cause serious systemic infections in humans and animals.^9,25 The genus Talaromyces has ~ 110 species, and several new species, including T. georgiensis, have been discovered in 2017–2018.^3,12 Herein, we report a case of arthritis caused by T. georgiensis in a dog.

A 6-y-old, 3.5-kg, spayed female Toy Poodle was presented with left forelimb lameness of 2-d duration. Radiography showed mild periosteal reaction of the left distal humerus. Serum biochemistry revealed an increased C-reactive protein level (CRP; 60.2 mg/L). The dog was diagnosed with arthritis and was treated with carprofen (2.2 mg/kg PO q12h for 1 mo; Rimadyl; Zoetis). One month later, during the second consultation, swelling of the elbow joint was observed. Radiography (Fig. 1A) and computed tomography (CT) showed osteolysis of the medial epicondyle of the left humerus. Arthritis (caused by autoimmune or infectious diseases) and neoplasia (such as synovial sarcoma) were differential diagnoses. Bacterial cultures, performed at Hoken Kagaku Laboratory (Kanagawa, Japan), using sheep blood agar, fungal cultures using Sabouraud agar to detect filamentous fungi and Cryptococcus spp., and CHROMagar candida medium (BBL; Becton Dickinson) to identify yeast-like organs of the synovial fluid, were negative. At reexamination 1 mo later, clinical signs had not resolved, and the left forelimb was amputated because of ongoing pain and muscle atrophy. The dog was subsequently treated with terbinafine (35 mg/kg PO q24h for 3 wk; TRF, Lamisil tablets 125 mg; Sun Pharmaceutical).

Figure 1.

Granulomatous polyarthritis caused by Talaromyces georgiensis in a dog. A. Lysis of the medial epicondyle of the left humerus (arrow) is present in this dorsal-ventral radiograph of the forelimbs. B. Sagittal section of formalin-fixed distal left humerus. The articular villi of the elbow joint are thickened, and the articular cartilage and cancellous bone of the distal humerus are ulcerated. C. In the inflamed synovial bursa, the cytoplasm of the macrophages is enlarged and foamy. H&E. Bar = 50 μm. D. Spherical fungal spores (3–10 μm diameter) and hyphae are present within the macrophages in the synovial bursa. Gomori methenamine silver stain. Bar = 50 μm.

The amputated forelimb was fixed in 10% formalin. Grossly, the articular villi of the elbow joint were markedly thickened (Fig. 1B). The articular cartilage surfaces of the distal humerus and proximal radius were extensively and partly irregular, and demonstrated erosion along with exposed subchondral bone (Fig. 1B); additionally, there was a 5-mm lytic lesion on the distal humerus. There were no ulcers or signs of trauma on the skin of the forelimb. Samples from the elbow joint were processed routinely and stained with hematoxylin and eosin, as well as Gomori methenamine silver (GMS) stain and periodic acid–Schiff (PAS) reaction. Histologically, severe granulomatous arthritis composed of infiltrates of numerous macrophages, lymphocytes, plasma cells, and a few neutrophils was observed in the articular villi of the elbow joint (Fig. 1C), underlying mild granulation tissue formation and edema. In addition, severe inflammation extended up to 3 mm into the bone marrow cavity of the distal humerus and proximal radius with mild granulation tissue; necrosis of the exposed distal humerus was observed. Most macrophages had cytoplasmic enlargement and contained abundant spherical yeast-like organisms 3–10 μm diameter, and 3-μm wide, septate, nonbranching hyphal-like structures. Both the yeast and hyphal-like organisms were positive with GMS (Fig. 1D) and PAS reaction. Fewer of these organisms were found extracellularly. Mild muscle atrophy around the joint was observed without inflammation. In the axillary lymph nodes, a few fungal-laden macrophages were found in the marginal sinus.

Four months after the amputation, the dog exhibited lameness of the right hindlimb. Radiography showed osteolysis in the medial aspect of the right stifle joint. Three weeks later, CT showed lysis and periosteal new bone production of the distal right femur medially, and mild enlargement of the right internal iliac lymph node. Tissues samples for histologic examination and fungal culture were taken from the distal right femur. Histologically, fungi with similar morphology to those observed previously in the left elbow joint were found within the macrophages.

Tissue samples were inoculated on a Sabouraud dextrose agar plate for 14 d at 28°C. Numerous flat, velvety, white and green powdery colonies were observed on the plate after 14 d incubation. On microscopic examination, septate, unbranching conidiophores and smooth-walled spherical conidia (2–3 μm diameter) were observed (Fig. 2).

Figure 2.

Septate, unbranching conidiophores and smooth-walled spherical conidia (2–3 μm diameter) are visible in fungal colonies cultured from the right distal femur in the dog. Bar = 20 μm.

Molecular identification of the fungus was conducted based on isolation of DNA genes using the colonies grown on the plate. The internal transcribed spacer (ITS) region of the DNA sample from the isolate was amplified using the universal fungal primers ITS5 (5′-GGAAGTAAAAGTCGTAACAAGC) and ITS4 (5′-TCCTCCGCTTATTGTAGC) ¹⁴ ; PCR amplification conditions and sequence analysis were performed as described previously. ² The β-tubulin primers benA-F (5′-AATTGGTGCCGCTTTCTGG) and benA-R (5′-AGTTGTCGGGACGGAATAG) were used to amplify DNA from Aspergillus species. ¹ Comparative nucleotide sequence analysis using the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that the ITS and β-tubulin sequences amplified from the isolate were 100% and 99% identical to type strain UTHSC D16-145^T of Talaromyces georgiensis GenBank accessions LT558967 and LT559084, respectively. ¹²

In vitro susceptibility testing of the fungus was performed using the E test and M38-A microdilution methods. ²² The antimicrobial susceptibility tests for this organism showed that the minimum inhibitory concentration (MIC) of itraconazole (ITZ), voriconazole, TRF, amphotericin B, and fluconazole was < 0.002 mg/L, 0.012 mg/L, 0.06 mg/L, 0.25 mg/L, and > 256 mg/L, respectively. The dog was prescribed a 6-mo course of ITZ (7.5 mg/kg PO q24h; 50-mg tablets; Nichi-Iko Pharmaceutical) and TRF (35 mg/kg PO q24h) after diagnosis of the right hindlimb lesion. After 6 mo of antifungal treatment, the dog’s clinical signs had not progressed.

The dog had granulomatous arthritis associated with numerous intracellular and extracellular yeast and hyphae-like organisms. These organisms were confirmed to be T. georgiensis by fungal cultures, sequencing, and searching BLAST. T. georgiensis was isolated from joint fluid in an animal in 2017 ¹² ; details, including animal species and pathogenesis, are unclear. T. georgiensis has not been reported previously to cause disease in humans or animals, to our knowledge. T. georgiensis was found in multiple arthritic joints of our case, suggesting that T. georgiensis was the cause of fungal arthritis.

Some species of Penicillium have been reclassified as Talaromyces because the emphasis in taxonomy has shifted from morphologic features to phylogenetics via DNA sequencing technology. ²⁴ With regard to Penicillium/Talaromyces spp., 3 cases of canine arthritis^25,26,29 and 2 cases of canine osteomyelitis^17,18 have been reported (Table 1). One of those cases was reported as a case of canine arthritis in the right cubital joint caused by a T. helicus infection. ²⁵ T. helicus and T. georgiensis belong to the Helici section of Talaromyces. ³ The histologic morphology of T. georgiensis is similar to the cytomorphology of T. helicus, hyphae are 3-μm wide, septate, unbranched, and present both intra- and extracellularly; round structures were observed in both cases. With regard to the T. helicus infection, fungi were also observed in the skin, subcutaneous tissues, lymph nodes, and the spleen at autopsy. However, the fungi in our case were localized to multiple joints and the lymph nodes. These differences may be the result of variation in the original site of infection, associated pathogenicities, antifungal drugs, or host immunity. The other 2 case reports of Penicillium/Talaromyces spp. infection involving joints of dogs were associated with the presence of P. purpureogenum (renamed T. purpureogenus ²⁸ ), which led to multiple lesions of diskospondylitis, ²⁹ and P. verruculosum (renamed T. verruculosus ²⁸ ), which led to osteomyelitis and arthritis of both hindlimbs. ²⁶ Canine osteomyelitis caused by Penicillium/Talaromyces spp., including P. canis ¹⁷ and T. verruculosus, ¹⁸ have been reported; the specific involvement of joints was not described in these cases.

Table 1.

Penicillium/Talaromyces spp. infection in joints and bones of dogs.

Case	Year	Fungus	New name	Age, sex, breed	Affected locations	Fungal morphology	Treatment	Outcome
1		T. georgiensis (this case)		6-y-old, SF, Toy Poodle	Left elbow joint, right stifle joint, lymph nodes	3-μm wide, septate, nonbranching hyphae, and round structures measuring up to 10 μm, intra- and extracellular	Left forelimb amputation, itraconazole, terbinafine	Improved
2	2019	T. helicus 25		8-y-old, CM, Labrador Retriever	Right elbow joint, skin, subcutaneous tissue, spleen, lymph nodes	2–4-μm wide, septate, nonbranching hyphae, and round structures 2–4-μm diameter, intra- and extracellular	Right thoracic limb amputation, amphotericin B, itraconazole	Euthanized
3	2006	P. purpureogenum 29	T. purpureogenus	4-y-old, F, German Shepherd	T10–T11, L2–L3, liver, spleen, lymph nodes	Extracellular, septate, branching hyphae	Itraconazole, enrofloxacin	Died
4	1990	P. verruculosum 26	T. verruculosus	3.5-y-old, CM, German Shepherd	Multiple bones and joints of both hindlimbs	2.5–3-μm wide, septate, branching hyphae, and round structures 6–8 μm diameter, extracellular	None	Euthanized
5	2011	P. verruculosum 18	T. verruculosus	7.5-y-old, SF, Collie	Left humerus, lymph nodes	3–5-μm wide, septate, branching hyphae, intra- and extracellular	Antifungal therapy	Euthanized
6	2014	P. canis 17		3-y-old, SF, Rhodesian Ridgeback	Right ilium	3–4-μm wide, septate, branching hyphae, and round structures 5–7 μm diameter, intra- and extracellular	Amphotericin B, terbinafine, ketoconazole	Improved

CM = castrated male; F = female; SF = spayed female

Although canine fungal arthritis is rare, the reported fungal causes include Aspergillus spp.,^{4,7,20,21,23,30} Cryptococcus spp.,^5,8,13,16 Blastomyces dermatitidis,^19,27 Candida guilliermondii, ⁶ Histoplasma capsulatum, ¹⁵ Paecilomyces sp., ¹⁰ and Sporothrix schenckii ¹¹ (Table 2).

Table 2.

Summary data of cases in dogs of fungal arthritis reported in the literature.

Fungus	Age, sex, breed	Joint	Extra-articular locations*	Treatment	Outcome
Aspergillus deflectus 23	4-y-old, F, German Shepherd	Right shoulder	Kidneys, adrenal glands, heart, lymph nodes	None	Euthanized
Aspergillus fumigatus 20	Adult, M, German Shepherd	Left elbow	None	None	Euthanized
Aspergillus terreus 4	3-y-old, F, German Shepherd	C2–C3, C7–T1, T2–L1, L2–L3, L5–L6	Pubis, kidney, heart, aorta	None	Euthanized
Aspergillus terreus 4	4-y-old, SF, German Shepherd	T12–T13, L5–L6	None	Ketoconazole	Euthanized
Aspergillus versicolor 30	2.5-y-old, CM, German Shepherd	T9–T10	Sternal, kidneys, liver, spleen, lymph nodes	None	Euthanized
Aspergillus sp. 7	4-y-old, SF, German Shepherd	T1–T2, T6–T7, T10–11, L3–6	None	Amoxicillin, clavulanic acid	Euthanized
Aspergillus sp. 21	2.5-y-old, SF, German Shepherd	T7–T8	Liver, lung, spleen, lymph nodes	None	Euthanized
Blastomyces dermatitidis 19	4-y-old, SF, mixed	Left stifle	None	Joint lavage, itraconazole	Improved
Blastomyces dermatitidis 27	6-mo-old, CM, Labrador Retriever	Right carpus	Lung	Joint lavage, itraconazole	Improved
Candida guilliermondii 6	5-y-old, M, Boxer	Left stifle	None	Joint lavage, fluconazole	Improved
Cryptococcus neoformans 5	1.5-y-old, M, German Shepherd	Left hock	Brain, lung, lymph nodes	None	Euthanized
Cryptococcus neoformans 16	1.5-y-old, SF, Cocker Spaniel	Both hocks, left carpus	Brain, liver, lung, spleen, lymph nodes	Nystatin	Euthanized
Cryptococcus neoformans var. grubii 13	4-y-old, F, Boxer	Right hock	Brain, lymph nodes	Itraconazole	Died
Cryptococcus sp. 8	4-y-old, M, mixed	Left elbow	None	None	Euthanized
Histoplasma capsulatum 15	3-y-old, CM, Labrador Retriever	Left carpal, left stifle, left hock	Left eye, lung, liver, lymph nodes	Ketoconazole, amphotericin B	Died
Paecilomyces sp. 10	5-y-old, F, mixed	T1–T2, T6–L7	Kidneys, mitral valve, aorta	Ketoconazole	Died
Sporothrix schenckii 11	5-y-old, SF, Labrador Retriever	Right hock	None	Ketoconazole	Improved

CM = castrated male; F = female; M = male; SF = spayed female.

*

Excluded bones around the affected joints.

Accurate identification of fungi by their histologic morphology alone is difficult. Molecular techniques such as DNA sequencing have made it possible to identify rare fungal species grown in culture or found in clinical specimens. Identification of the fungal agent is important for selecting appropriate antifungal therapy.

The most common routes of fungal infection are invasion from inhalation of spores, traumatic inoculation of spores, or hematogenous invasion by septicemia. In our case, no obvious immunologic abnormalities, or concurrent disease such as pneumonia or septicemia, were identified, thus it remains unclear how the fungus became widely disseminated.