A Case of Cutaneous Sterile Pyogranuloma/Granuloma Syndrome in a Maltese

Introduction

Cutaneous sterile pyogranuloma/granuloma syndrome (SPGS) is a skin disease that is uncommon in dogs and extremely rare in cats.^1–4 In the literature, SPGS is also termed idiopathic periadnexal multinodular granulomatous dermatitis.² No age or gender predisposition is reported; however, boxers, golden retrievers, collies, Great Danes, and Weimaraners may be predisposed, and male dogs seem to be affected at a higher frequency.^1–5 Characteristic skin lesions are usually observed on the bridge of the nose, muzzle, periocular region, pinnae, and paws.^1–5 These lesions comprise dermal papules and nodules, which are multiple, well demarcated, firm, painless, and nonpruritic.^1–5 They may become alopecic and ulcerated and may get secondarily infected, particularly on the paws.^1–5 Although systemic signs such as lymphadenopathy and hypercalcemia have been reported in dogs and cats, the affected animals are typically healthy.^1–7

The diagnosis of cutaneous SPGS is challenging and can be made only after ruling out other granulomatous and pyogranulomatous skin diseases.^1–4 According to Santoro et al. (2008), the etiology of granulomatous and pyogranulomatous skin diseases can be divided into two major groups. The first group is infectious diseases caused by protozoans, mycobacteria, pathologic dimorphic fungi, etc. and noninfectious diseases with a known etiology such as a foreign body, hair, sebum, etc. The second group includes unknown etiologies, such as idiopathic “sterile” dermatitis.⁴ Histopathologically, those criteria were also based on visible infectious and known etiological agents and etiologic agents not detectable by analytical methods and possible immune-mediated (idiopathic) mechanisms. Some organisms such as Mycobacterium spp. and Leishmania spp. are not easily detected by histopathology; therefore, polymerase chain reaction (PCR) analysis is required.^4,7,8 A diagnosis of cutaneous SPGS can be made after ruling out noninfectious granulomas of unknown etiologies, such as reactive histiocytosis, juvenile sterile granulomatous dermatitis and lymphadenitis, cutaneous xanthoma, and canine sarcoidosis.^1–4

Herein, the successful long-term treatment of a dog presenting with cutaneous SPGS characterized by generalized ulcerative dermatitis, systemic signs of mild anemia, and an increased C-reactive protein (CRP) level is described. Cutaneous SPGS was diagnosed by histopathology, negative staining, and PCR for Mycobacterium spp.

Case Report

An indoor 3 yr old castrated male Maltese weighing 4.2 kg was referred to the Veterinary Teaching Hospital, Azabu University, with generalized ulcerative dermatitis. Initially, the skin lesions occurred on the dog’s head, identified by mild scales. The lesions progressed to crusting and multifocal ulcers, which spread to the head, dorsal and ventral trunk, and limbs over a period of 8 mo. Prior to being referred, the dog was administered systemic antibacterial and antifungal drugs [30 mg/kg cephalexin per os (PO) q 12 hr and 6 mg/kg ketoconazole PO q 12 hr, respectively] for 4 wk; however, the dog was unresponsive. Physical and dermatological examinations, bacterial culture with antimicrobial susceptibility testing, fungal culture, blood tests, diagnostic imaging, urinalysis, arthrocentesis, skin biopsy, and histopathological examinations were subsequently performed at the time of referral.

On physical examination, the dog presented with severe pain, mild pruritus of the skin, and a body temperature of 39°C. Bilateral lymphadenopathy of the superficial cervical and popliteal lymph nodes was observed. Skin lesions comprised alopecia and erythema with epidermal detachment and multifocal ulcers with crusting. Although haired skin on the top of the muzzle remained, demarcated alopecic skin lesions were observed on the dog’s face, including the periocular, periauricular regions, and the planum nasale from the top of the muzzle to the head. Other affected areas were the dorsal trunk, extending from the neck to the front of the tail; the ventral trunk extending from the anterior chest; and the axilla to the inguinal region, limbs, paws, and perianal region (Figure 1). Impression smears were obtained from the ulcerative skin lesions of the dorsal and ventral regions and stained by the Wright’s-Giemsa method. Cytology revealed a pyogranulomatous inflammation comprising macrophages and nondegenerate neutrophils; however, acantholytic keratinocytes, bacteria such as cocci and bacilli, and Malassezia spp. were not apparent. Infection with Demodex canis was ruled out by skin scraping and hair plucking, and sarcoptic mange was ruled out by only mild pruritus of the skin.

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Hematologic analysis revealed results within reference ranges, except for a slight decrease in the red blood cell count (3.92 × 10¹²/L; reference range, 5.5–8.5 × 10¹²/L) and packed cell volume (0.31%; reference range,) and a remarkable increase in CRP level (142.86 nmol/L; reference range 0.37–0.55, < 9.52 nmol/L) as described in Table 1. After severe inflammation was suspected in the dog, CRP level was monitored using a laser nephelometric immunoassay system^a for dogs.⁹ Serum protein electrophoresis revealed an increased α₂-globulin fraction level, but did not indicate gammopathy.

TABLE 1 Complete Blood Cell Count, Serum Biochemical Profile, and Urinalysis Results on the Day of Referral

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Differential diagnoses from the findings at the first visit were both aerobic and anaerobic bacteria, including Nocardia and Actinomyces spp.; deep and intermediate fungi; dermatophytosis; pemphigus foliaceus; pemphigus vulgaris; erythematosus paraneoplastic pemphigus; discoid lupus erythematosus; systemic lupus erythematosus; cutaneous lymphoma; erythema multiforme; cutaneous vasculitis; toxic epidermal necrolysis; hepatocutaneous syndrome; and idiopathic ulcerative dermatosis.

Antimicrobial susceptibility testing was conducted at Hoken Kagaku Laboratory (Kanagawa, Japan) using the Clinical Laboratory Standards Institute method. Aerobic bacterial culture from a swab of a dorsal ulcerative skin lesion using sheep blood agar was negative. Although the association of anaerobic bacteria could not be ruled out, anaerobic culture was not performed because the ulcerative lesion was aerobically exposed on the superficial epidermis. Fungal culture performed and analyzed at Hoken Kagaku Laboratory from the same skin lesion was negative. Sabouraud agar, which is used for detection of dermatophytes, yeast-like organisms, and Cryptococcus spp., was used for the culture. Because specific bacterial and fungal features were not identified by the impression smears, macerated tissue culture and specialized culture were not performed.

Thoracic and abdominal radiographs and ultrasonography did not reveal any apparent abnormalities. Dermatoses associated with systemic diseases such as paraneoplastic pemphigus and hepatocutaneous syndrome was ruled out because none of findings indicated either a tumor mass or a remarkable abnormality of the liver parenchyma. To examine the possibility of systemic lupus erythematosus, urinalysis (Table 1), and arthrocentesis of synovial fluid from the left knee joint were performed. Urine test strips were positive for protein (2+) and negative to occasionally positive leukocytes, nitrate, urobilinogen, blood, ketones, bilirubin, and glucose. Zero to occasional red blood cells, white blood cells, and transitional cells were seen, but no crystals or casts were detected by urinary sediment examination. The synovial fluid was clear, colorless, and extremely viscous. Cytology revealed a few large and small mononuclear inflammatory cells, which was not suggestive of arthropathy.

Specimens were collected using a 4 mm punch biopsy from skin lesions on the dog’s head and dorsal trunk. Histopathology revealed a nodular to diffuse dermatitis characterized by intense lymphohistiocytic infiltration and fibrosis involving the superficial dermis and subcutis (Figure 2). The adnexa, such as the hair follicles and sebaceous glands, were obscured by fibrosis. Deposition of autoantibodies in the tissue was not detected by immunofluorescence using fluorescein isothiocyanate-labeled anti-dog immunoglobulin (Ig) G antibodies^b (diluted 1:800). Based on the histopathological evaluation, pemphigus foliaceus, pemphigus vulgaris, and systemic and discoid lupus erythematosus, cutaneous lymphoma, erythema multiforme, cutaneous vasculitis, toxic epidermal necrolysis, and idiopathic ulcerative dermatosis were ruled out. Histopathological analysis revealed the presence of nodular granuloma, which suggested a deep pyoderma and fungal infection by an antibiotic-resistant microorganism, atypical mycobacteriosis (canine leproid granuloma syndrome), and cutaneous SPGS. Cutaneous leishmaniasis was ruled out because it is not endogenous to Japan and the dog has never been outside Japan. Periodic acid-Schiff, Ziehl-Neelsen, Grocott’s methenamine silver, and gram staining were used, but failed to detect any etiologic agent in the tissue specimens.

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Using the primers described by Cornegliani et al. (2005), PCR was performed to detect Mycobacterium spp.⁸ DNA extraction from paraffin-embedded tissues was conducted using a commercial kit^c. Five microliters of the extracted DNA solution were used for the nested PCR amplification of a 16S rRNA fragment with a DNA polymerase^d using a gradient thermal cycler^e. The external primers (FO16S, 5′-GATAAGCCTGGGAAACTGGGTC-3′; RO16S, 5′-TTCTCCACCTACCGTCAATCCG-3′) were used to amplify a 344-base pair fragment and the internal primers (F116S, 5′-CATGTCTTGTGGTGGAAAGCG-3′; RI16S, 5′-TACCGTCAATCCGAGAGAACCC-3′) were used to amplify a 288-base pair fragment. The first and second PCR amplification consisted of one cycle at 98°C for 3 min, followed by 35 cycles of denaturation (98°C for 30 sec), annealing (65.7°C for 30 sec), and extension (72°C for 30 sec). The PCR products were separated by 2% agarose gel electrophoresis, stained with ethidium bromide, and visualized using an UV transilluminator^f. DNA was extracted from a BCG vaccine strain^g and used as a positive control. The PCR results were negative, and mycobacteriosis was ruled out in the present case.

Clinical findings (age of onset and breed), histopathological findings, and diagnostic imaging results ruled out the other noninfectious granulomas of unknown etiologies; therefore, a diagnosis of cutaneous SPGS was considered most suitable for the present case. Consequently, prednisolone^h (1 mg/kg PO q 12 hr) and fosfomycin Ca hydrateⁱ (25 mg/kg PO q 12 hr) therapy was initiated. In addition, as a supportive therapy, famotidine^j (1 mg/kg PO q 12 hr) and ursodeoxycholic acid^k (10 mg/kg PO q 12 hr) was administered for protection against the adverse effects of prednisolone. The treatment of skin barrier restoration included 0.5% vitamin A oil^l and a topical corticosteroid preparation comprising 0.12% betamethasone valerate^m and 0.3% heparinoidⁿ. One wk after the initiation of therapy (day 7), the ulcerative dermatitis improved and CRP level decreased (4.76 nmol/L). After 1 mo (28 days), the dog was free of grossly visible ulcerative skin lesions (Figures 3A–C) and the packed cell volume increased to 43%. Subsequently, the dose of prednisolone was tapered by 0.5 mg/kg BW at 2 wk intervals and administration of azathioprine^o (1 mg/kg PO q 24 hr) was initiated. After decreasing the dose of prednisolone to 0.5 mg/kg q 48 hr (day 82), it was further decreased to q 72 hr for 2 mo (day 110). Following the cessation of prednisolone on day 138, mild skin lesions with crusting and erosion were observed on the dorsum of the neck on day 166 (Figures 3D, E). Those relapses were treated with prednisolone, and the dosage of azathioprine was decreased to 1 mg/kg q 48 hr. Maintenance doses of prednisolone (0.5 mg/kg q 48 hr) and azathioprine (1 mg/kg q 48 hr) were selected on day 197; however, the owner had administered a single dose of prednisolone (0.5 mg/kg) 1–2 times/wk according to the owner’s judgment, although the dog received azathioprine as scheduled. At a 3 mo routine follow-up examination (day 278), the dog exhibited mild skin lesions of erythema and crusting on the limbs and dorsal and ventral trunk. A fungal culture using a dermatophyte test agar medium was negative. The owner was reminded to continue prednisolone. The skin symptoms did not become aggravated and remained unchanged for at least 9 mo. However, on day 537 during a routine follow-up examination, the owner reported an apparent recurrence of pruritus and crusting on the dog’s face and back within 2 wk after discontinuing prednisolone and azathioprine. The recurrent lesions were treated with prednisolone and azathioprine that were prescribed by the referring veterinarian. No hair regrowth was observed during the course of the treatment (day 624).

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Discussion

At the time of referral, the study authors considered a diagnosis of pustular skin disease; however, based on the histopathological findings of granulomatous dermatitis, that diagnosis was changed to granulomatous and pyogranulomatous skin disease. Based on the criteria described by Santoro et al. (2008), a diagnosis of cutaneous SPGS was made.⁴

The etiology and pathogenesis of cutaneous SPGS is unknown.^1–8 The absence of infectious agents and foreign materials together with a good response to systemic glucocorticoids suggested the involvement of immune-mediated mechanisms.^1–7 In the present case, neither causative organisms nor foreign bodies were detected by culture, impression smears, special histopathological staining, or PCR for Mycobacterium spp. Because Leishmania spp. is not endogenous to Japan, diagnoses were only made for Mycobacterium spp.^4,8 Although potentially infectious bacteria, such as antimicrobial-resistant bacteria, anaerobic bacteria, and bacteria requiring special culture methods, were not ruled out, the authors attempted to limit the possibility of infection using fosfomycin. Fosfomycin is a broad-spectrum antibiotic for gram-positive and gram-negative bacteria and is well distributed in tissues with clinically relevant concentrations in the serum, kidneys, bladder wall, prostate, lungs, inflamed tissues, bone, cerebrospinal fluid, abscess fluid, and heart valves.¹⁰ The efficacy of fosfomycin against cephalexin-resistant Staphylococcus spp. isolated from a patient with pyoderma and otitis externa has been reported in Japan.¹¹ In the present case, systemic inflammation was indicated by fever, an increased CRP level, and lymphadenopathy. The dog was also treated with immunosuppressive drugs, including prednisolone and azathioprine, although recurrences of skin lesions were observed when both drugs were discontinued. Those findings suggested that systemic inflammation, which was treated with prednisolone and azathioprine, was associated with the pathogenesis in the present case.

Hair regrowth was not observed even after successful treatment with prednisolone and azathioprine, possibly due to the presence of extensive fibrosis involving the superficial dermis and subcutis at the site of adnexa. The dermal papilla and the bulge region, where arrector pili muscles insert into hair follicles, are considered to contain stem cells capable of hair and skin regeneration.^12,13 The presence of fibrosis suggested severe inflammation that possibly destroyed the hair follicles and sebaceous glands at the affected sites.

In the present case, lymphohistiocytic infiltrations were observed by histopathology; however, deposition of the IgG antibody was not detected using immunofluorescence. It has been proposed that cutaneous SPGS is associated with immune dysfunction in response to chronic antigenic stimulation.¹ The absence of IgG autoantibodies suggested that the pathogenesis of the diseases was not associated with humoral-mediated immunity (as observed in pemphigus complex) but with cell-mediated immunity, in which effecter cells comprise macrophages and lymphocytes, as observed in the present case. The examination of surface cluster of differentiation antigens and major histocompatibility complex molecules on infiltrated cells could provide clues to elucidate the immune mechanisms involved in cutaneous SPGS.

Conclusion

Cutaneous SPGS is an uncommon skin disease in dogs.^1–4 The affected animals are typically healthy, but a few exhibit systemic signs.^1–7 This report documented a case of SPGS diagnosed by histopathological findings of a nodular to diffuse granulomatous dermatitis with intense lymphohistiocytic infiltration, negative staining for causative organisms, and PCR for Mycobacterium spp. The dog presented with generalized ulcerative dermatitis and systemic signs of mild anemia and an increased CRP level. Successful treatment was achieved by administration of immunosuppressive drugs (prednisolone and azathioprine) for at least 20 mo. Recurrence of skin lesions after cessation of prednisolone and/or azathioprine was observed, suggesting that long-term management using immunosuppressive drugs may be required if the dog exhibits severe systemic signs of cutaneous SPGS.