由美洲瓶霉菌引起的犬暗色丝孢霉病

王帆

Phaeohyphomycosis caused by Phialophora americana in a dog

由美洲瓶霉菌引起的犬暗色丝孢霉病

翻译：王帆

Abstract

摘要

Phaeohyphomycosis was diagnosed in a 6-year-old, male castrated Dachshund on immunosuppressive treatment. The fungus was identified by culture and PCR as Phialophora americana. This is the first reported case of infection with this pathogen in a dog. The infection was successfully managed medically, without surgical intervention.

一只6岁、已去势雄性，接受免疫抑制治疗的腊肠犬诊断为暗色丝孢霉病。经真菌培养和PCR鉴定，该真菌为美洲瓶霉菌。这是首例报告的犬感染这种病原体的病例。感染经药物治疗成功得以控制，没有进行外科手术。

INTRODUCTION

介绍

Dematiaceous fungi, also called melanised fungi or phaeohyphomycetes, are a heterogeneous group of organisms belonging to >60 genera. They are ubiquitous in the environment and typically nonpathogenic. The term “phaeohyphomycosis” describes infections caused by melanised fungi, occurring in immunocompetent or immunosuppressed individuals.

暗色真菌又称黑化真菌或暗色丝孢霉菌，是一种＞60属的异质性的生物类群。它们在环境中无处不在，而且通常不会致病。“暗色丝孢霉病”一词描述由黑化真菌引起的感染，发生在免疫正常或免疫抑制的个体中。

Several clinical syndromes of phaeohyphomycosis are distinguished, with a wide range of organ manifestations (skin, subcutis, cornea, lungs, brain). In addition, allergic and disseminated disease have been described. Current therapeutic recommendations in human and veterinary medicine are based on clinical experience or individual case reports, and involve a combination of surgical and medical treatment. The prognosis usually is guarded.

暗色丝孢霉病有多种临床综合征，具有广泛的器官表现(皮肤、皮下组织、角膜、肺、脑)。此外，过敏症和弥散性疾病也有描述。目前人类和动物医学的治疗建议是基于临床经验或个案报告，涉及外科和内科治疗的结合。预后通常是保守的。

Phialophora americana has been isolated mainly from the environment, and cases of infection by this pathogen in humans have been reported recently in China and Mexico. To the best of the authors' knowledge, there are to date no reported cases of infection with P. americana in the veterinary literature. Herein we report on clinicopathological features and a successful nonsurgical treatment approach of a case of phaeohyphomycosis caused by P. americana in a dog.

美洲瓶霉菌主要从环境中分离，最近在中国和墨西哥报告了人类感染这种病原体的病例。据作者所知，迄今为止在兽医文献中没有美洲瓶霉菌感染病例的报告。在此，我们报告一例由美洲瓶霉菌引起的犬暗色丝孢霉病的临床病理特征和非手术成功治疗方法。

CASE REPORT

病例报告

A 6-year-old, male neutered Dachshund was presented with multiple, multifocal and progressive skin lesions. At the time of presentation, the dog had received immunosuppressive therapy for meningoencephalitis of unknown origin. Medications given up to the day of presentation included prednisolone 0.5mg/kg every other day (Prednisolone Streuli, Streuli Pharma AG; Uznach, Switzerland), ciclosporin 5mg/kg once daily (Atopica, Elanco Animal Health; Hook, UK) and amoxicillinclavulanic acid 20mg/kg twice daily (Clavaseptin, Vetoquinol; Paris, France). Prednisolone had been administered for three months and ciclosporin for two months before presentation. Antimicrobial therapy had been started one week before presentation, owing to a suspicion of pyoderma.

一只6岁、雄性已去势腊肠犬因出现多灶性、多局灶性和渐进性皮肤损病变就诊。就诊期间犬已经接受免疫抑制剂，治疗不明原因的脑膜脑炎。直到就诊当天以前，用药史包括泼尼松龙0.5mg/kg，隔日一次，环孢素5mg/kg，每日1次和阿莫西林克拉维酸20mg/kg，每日两次。就诊前已用泼尼松龙3个月，环孢素2个月。由于怀疑有脓皮病，在就诊前一周开始抗生素治疗。

On physical examination, multiple intradermal and ulcerated nodules were noted on the scrotum (Figure 1a).Additional lesions also were present on the dorsum, and appeared as focal, circular alopecia with erosive crusts (Figure 1b). The affected skin was thickened and the diameter of lesions ranged from 1 to 2cm. A similar lesion was present on the dorsal aspect of the nose. Cytological examination of an impression smear acquired from an active lesion revealed inflammation with several fungal elements (hyphae; Figure 2). Punch biopsies of the skin lesions on the dorsum were taken for histopathological examination, and a skin scraping was submitted for fungal culture.

体格检查发现阴囊多发皮内和溃疡性结节(图1a)。背部也出现了其他病变，并表现为局灶性圆形脱毛伴糜烂性结痂(图1b)。患病皮肤增厚，病变直径1-2cm。鼻背侧也有类似的病变。活跃病变皮肤压片采样细胞学检查，显示多种真菌元素(菌丝;图2)。对背侧皮肤病变打孔活检进行组织病理学检查，以及皮肤刮片采样进行真菌培养。

Histopathological features included high-grade, multifocal to confluent, pyogranulomatous dermatitis with intralesional evidence of fungal elements (Fig. 3). The fungal elements were spherical, 10–20μm in diameter, sprouting on a broad base (and without endosporulation) and failed to stain with mucicarmine. The fungal culture was positive for Phialophora sp. and DNA sequencing was performed for complete species identification. Fungal genomic DNA was isolated using a DNeasy Plant Mini Kit (Qiagen AG; Hombrechtikon, Switzerland) according to the manufacturer's protocol, and part of the 28S ribosomal DNA subsequently was amplified by PCR using the universal primers LSU1 (5′-GATAGCGMACAAGTAGAGTG-3′) and LSU2 (5′-GTCCGTGTTTCAAGACGGG-3′). A 300bp amplicon was detected in agarose gels after electrophoresis. The sequences of the PCR product were found by Blast analysis to be 100% identical to the sequence MH870708 deposited for 28S ribosomal DNA of P. americana strain 400.67.

组织病理学特征包括高级别、多灶性、融合性脓性肉芽肿性皮炎，病灶内可见真菌元素(图3)。真菌元素呈球形，直径为10-20μm，在较宽的基底发芽(且无内生孢子)，粘蛋白胭脂红染色不着染。真菌培养结果为瓶霉菌属阳性，并进行DNA测序进行完整菌种鉴定。采用DNeasy Plant Mini试剂盒按说明分离真菌基因组DNA，随后，使用通用引物LSU1 (5′-GATAGCGMACAAGTAGAGTG-3′)和LSU2 (5′-GTCCGTGTTTCAAGACGGG-3′)通过PCR扩增部分28S核糖体DNA。电泳后在琼脂糖凝胶中检测一个300bp的扩增子。经Blast分析，PCR产物序列与美洲甁霉菌400.67株28S核糖体DNA MH870708序列完全一致。

Complete haematology and serum chemistry, chest radiographs and abdominal ultrasound were performed to rule out a systemic infection. All results were unremarkable. Treatment with topical clotrimazole ointment (Fungotox Crème, Mepha Pharma AG; Basel, Switzerland) twice daily and systemic itraconazole 10 mg/kg once daily per os (Sporanox, JanssenCilag AG; Zug, Switzerland) was initiated. In order to accelerate the treatment success, immunosuppressive medications were slowly tapered. Surgical removal of the nodules on the scrotum was suggested (castration), yet the owner declined this option.

全血细胞计数和血清生化检查、胸片和腹部超声检查以排除系统性感染。所有结果都未见异常。治疗外用克霉唑软膏，每日2次，全身使用伊曲康唑10 mg/kg，每日1次口服。为了加快治疗的成功，免疫抑制剂逐渐减量。建议手术切除阴囊上的小结节(去势)，但宠主拒绝了这个选择。

The dog was presented for the first follow-up examination two weeks after anti-fungal therapy was initiated. The nodules on the scrotum and the lesions on the back were reduced by approximately 50% in size and the crust on the nose had resolved. Fungal elements were no longer visible cytologically. The dog was presented again following an additional four weeks of therapy, and all skin lesions had resolved completely. Itraconazole (10mg/kg) was tapered to every third day and continued for one more month. Prednisolone (0.5mg/kg) and ciclosporin (5mg/kg) also were tapered to every third day. At the time of publication, two years after the diagnosis of phaeohyphomycosis, the dog remained stable with no relapse of fungal skin infection.

抗真菌治疗两周后，对患犬进行首次复诊检查。阴囊上的结节和背部的病变缩小了大约50%，鼻部结痂也消失了。细胞学检查未见真菌元素。持续治疗4周后，患犬再次复诊，所有的皮肤病变已完全恢复。伊曲康唑(10mg/kg)逐渐量至每3天一次，持续1个月。泼尼松龙(0.5mg/kg)和环孢素(5mg/kg)也逐渐减量到每3天一次。在本文发表时，在确诊暗色丝孢霉病两年后，该犬保持稳定，没有复发真菌性皮肤感染。

DISCUSSION

讨论

To the best of the authors' knowledge, this is the first case report of cutaneous phaeohyphomycosis caused by P. americana in a dog. Phialophora sp. are most prevalent in China and other parts of Asia, while in other parts of the world, Alternaria sp. are the most common cause of phaeohyphomycosis. Phialophora americana has been isolated mostly from the environment and causes subcutaneous infections of humans. In veterinary medicine, four reports have described infections by Phialophora sp. in cats with solitary, subcutaneous lesions. These cases were managed by surgical resection alone. One cat experienced a relapse while two others did not, and in the fourth case, no follow-up was available. Cases of phaeohyphomycosis reported in dogs describe mostly systemic infections with poor outcomes. Most of the cases in which remission was achieved involved surgical excisiony and medical treatment over several months.

据作者所知，这是第一例由美洲瓶霉菌引起的犬皮肤暗色丝孢霉病的报告。瓶霉菌属在中国和亚洲其他地区最普遍，但在世界其他地区，链格孢霉属是最常见的暗色丝孢霉病病因。美洲瓶霉菌主要从环境中分离，可引起人皮下组织感染。在兽医中，已报道四例猫感染瓶霉菌，表现为独立的、皮下组织病变。这些病例均为单纯手术切除治疗。一只猫复发，另两只没有复发，在第四个病例没有随访。报告的犬暗色丝孢霉病病例大多为全身性感染，预后较差。大多数病例通过手术切除和数月的药物治疗得以缓解。

Cutaneous phaeohyphomycosis typically manifests as single or multifocal nodules that may ulcerate or develop draining tracts. Lesions described for the present case thus are not typical of phaeohyphomycosis. However, two cases with similar lesions, such as alopecia, crusts and papules, have been reported. Another interesting feature of the present case was the presence of fungal elements in the hair follicle. The fungal elements were positive on Fontana–Masson stain (Figure 3), which, together with the fungal culture result, allowed us to exclude the presence of a concomitant dermatophyte infection. Moreover, the presence of intrafollicular pigmented fungal elements in phaeohyphomycosis already has been reported. In these cases, it was hypothesised that the infection originated in the cornified skin layers and that hair follicles were the portal of entry for dermal entry, which also could have been the case herein.

皮肤暗色丝孢霉病典型表现为单一或多灶性结节，可能溃疡或发展为瘘道。因此，本病例所描述的病变不是典型的暗色丝孢霉病。然而，已报道有2例病例有类似的病变，如脱毛、结痂和丘疹。本病例另一个有趣的特征是毛囊中存在真菌元素。真菌元素氨银液染色阳性(图3)，这与真菌培养结果一起，使我们能够排除并发皮肤癣菌感染的存在。此外，在暗色丝孢霉病中已有毛囊内存在色素性真菌元素报道。在这些病例中，假设感染起源于皮肤角质层，毛囊是进入真皮层的入口，这也可能表现为本例的情况。

At present, management of cases with infections resulting from dematiaceous fungi is based mainly on data from case reports and textbooks, and larger studies are lacking. The recommended therapy in cases of subcutaneous phaeohyphomycosis is in fact aggressive surgical excision, followed by systemic antifungal therapy for at least three months. In our case, the owner rejected surgical treatment. Contrary to expectations, however, the lesions healed within a few weeks with medical treatment only. The reduction of immunosuppressive drugs may have played an important role in the resolution of the skin lesions.

目前，暗色真菌感染病例的管理主要基于病例报告和教科书的信息，缺乏更大规模的研究。皮下组织暗色丝孢霉病的治疗建议实际上是积极的手术切除，然后进行至少三个月的全身抗真菌治疗。在我们的案例中，宠主拒绝手术治疗。然而，与预期相反的是，病变仅通过药物治疗，几周内就痊愈了。免疫抑制剂的减量可能在皮肤病变的恢复中发挥了重要作用。

Correct identification of the organism is an important aspect in the management of a case of phaeohyphomycosis. An example of this is the fact that some dematiaceous fungi appear to be neurotropic (e.g. Cladophialophora sp.), and thus the treatment plan requires the use of antifungal drugs that reach the central nervous system. All reported cases of Phialophora sp. infections in animals have been located in the skin. One might therefore speculate that in the case of infection by this species, systemic dissemination is less likely and a more conservative therapeutic approach may – as in our case – be effective. Obviously, this observation should be approached with caution, and further large-scale studies are needed.

正确识别微生物是管理暗色丝孢霉病的重点。一个例子是，一些暗色真菌似乎是嗜神经的(例如斑替支孢瓶霉属)，因此治疗方案要求使用能到达中枢神经系统的抗真菌药物。所有报告的动物感染瓶霉菌的病例病变都局限于皮肤。因此，人们可以推测，在这种菌种感染的病例中，全身性传播的可能性较小，就本病例而言，更保守的治疗方法可能是有效的。显然，这一观察结果应该谨慎对待，并需要进一步的大规模研究。

In conclusion, this case report highlights the importance of fungal species identification for correct treatment and precise case reporting in rare diseases. It could be shown that, contrary to previous reports, medical treatment alone may result in rapid and effective remission of clinical signs in dogs with cutaneous phaeohyphomycosis, without the need for surgical intervention.

总之，本病例报告强调了真菌菌种鉴定对罕见病的正确治疗和准确病例报告的重要性。这可以表明，与以往的报道相反，单纯的药物治疗皮肤暗色丝孢霉病可能能达到临床症状迅速恢复和有效缓解，而不需要手术干预。

FIGURE 1 Lesions caused by opportunistic infection from Phialophora americana in a Dachshund after immunosuppressive therapy (a) Multiple, ulcerated nodules on the scrotum, and (b) crusted and alopecic lesion on the lateral aspect of the thigh

图1腊肠犬经免疫抑制治疗后，导致感染条件致病菌美洲瓶霉菌，引起的病变(a)阴囊多发性溃疡性结节，(b)大腿外侧结痂和脱毛病变。

FIGURE 2 Photomicrographs of a cytological specimen from one lesion on a Dachshund presenting with phaeohyphomycosis Hyphae (black arrow) and neutrophils (grey arrow) are visible; DiffQuik, ×1,000

图2一只腊肠犬的一个病变的细胞学检查样本的显微照片，可见暗色丝孢霉病的菌丝(黑色箭头)和中性粒细胞(灰色箭头);DiffQuik，1000倍

FIGURE 3 Histological sections of skin biopsy specimens from a Dachshund presenting with phaeohyphomycosis

Fungal elements present in the dermal tissue are highlighted by black arrows, and those in the hair follicles by grey arrows: (a) Periodic acid Schiff; 200x (b) Fontana-Masson; 100x. Melanin is visible within the cell walls of the fungi

图3一只患暗色丝孢霉病腊肠犬的皮肤活检标本的组织学切片。

可见真皮层组织中的真菌元素用黑色箭头指出，毛囊中的真菌元素用灰色箭头指出:(a)PAS染色;200倍 (b)氨银液染色;100倍。在真菌的细胞壁内可以看到黑色素。