犬猫马拉色菌性皮炎的生物学，诊断和治疗-2020（6-11）

王帆

Biology, diagnosis and treatment of Malassezia dermatitis in dogs and cats

犬猫马拉色菌性皮炎的生物学，诊断和治疗-2020

Clinical Consensus Guidelines of the World Association for Veterinary Dermatology

世界兽医皮肤病学会临床共识指南

Ross Bond , Daniel O. Morris, Jacques Guillot, Emmanuel J. Bensignor , David Robson, Kenneth V. Mason, Rui Kano and Peter B. Hill

翻译：辛蕾 校对：王帆

6. Pathogenesis: immunological responses to Malassezia yeasts and their clinical relevance in diagnosis/therapy

发病机理：对马拉色菌的免疫应答及其临床诊断/治疗的相关性

The presence of Malassezia organisms on the skin, both in normal and excessive numbers, is known to activate the skin immune system. There is now compelling evidence that Malassezia antigens can stimulate innate, antibody and cell mediated immune responses, as well as triggering hypersensitivity reactions.Although this immune response may offer a degree of protection in certain circumstances, it may also be harmful. In animals in which an overgrowth of organisms has occurred, or in individuals that are predisposed to allergic sensitization, the ensuing inflammatory response can lead to clinical signs such as dermatitis and pruritus. This section comprises a comparative review of human and dog studies; publications on immune reactivity to Malassezia species in cats appear to be lacking.

已知在皮肤上存在正常数量和过量的马拉色菌，都能激活皮肤的免疫系统。现在有令人信服的证据显示，马拉色菌抗原可以激发先天的，抗体和细胞介导的免疫反应，也可引发超敏反应。尽管这种免疫反应在某些情况下可能会提供一定程度的保护，但它也可能有害处。在有机体过度增殖的动物中，或者在易过敏的个体中，随之而来的炎症反应可致临床症状，比如皮炎和瘙痒。这部分包括了对人和犬研究的比较回顾；似乎缺乏有关猫与马拉色菌属的免疫反应性有关的出版物。

6.1 Activation of keratinocytes

6.1角质形成细胞的激活

The initial interplay between Malassezia organisms and the skin immune system is likely to take place in the epidermis.It has been demonstrated that application of M. pachydermatis suspensions on healthy dog skin can induce skin lesions similar to those observed in naturally occurring Malassezia dermatitis.This indicates that Malassezia cell surface markers or metabolic products derived from the yeast may be able to directly damage the skin or induce pathogenic effects by activating the skin immune system.In order for this to happen, antigens or allergens produced or expressed by Malassezia spp. would need to penetrate the stratum corneum in order to be recognised by Langerhans cells or keratinocytes. These would then act as antigen presenting cells able to sensitise and then activate the T lymphocyte population. An impaired barrier function, such as is seen in canine and human atopic dermatitis, is likely to facilitate transepidermal allergen penetration. Activation of keratinocytes by Malassezia is suggested by studies in humans which demonstrated that M. furfur could invade keratinocytes and resist phagolysosome fusion.Furthermore, M. sympodialis produces extracellular vesicles, enriched with the allergens Mala s 1 and s 7, that bind actively to and are internalised by human kertinocytes, potentially promoting sensitisation and maintenance of inflammation.

马拉色菌与皮肤免疫系统之间的初始相互作用可能发生在表皮层。已经证明了健康犬皮肤上的厚皮马拉色菌悬浊液可诱导皮肤病变，类似于自然发生的马拉色菌皮炎。这表明了马拉色菌细胞表面标志物或源自酵母菌的代谢产物可能直接损伤皮肤或通过激活皮肤免疫系统来诱发疾病。为了达到这一目的，马拉色菌产生或表达的抗原或过敏原，需要穿透角质层才能被郎格罕细胞或角质形成细胞识别。这些充当了抗原递呈细胞使其致敏，然后激活T淋巴细胞群体。例如犬和人的特应性皮炎，皮肤屏障功能受损会促进表皮过敏原渗透。马拉色菌对角质形成细胞的激活，在人体研究上表明糠秕马拉色菌可以侵入角质形成细胞，并抵抗吞噬小体融合。此外，合轴马拉色菌产生细胞外囊泡，富含与马拉色S1和S7结合的过敏原，对人角质形成细胞具有积极作用并被其内在化，从而潜在地促进了致敏化和炎症的维持。

Keratinocytes recognise Malassezia antigens via Toll-like receptors, although they, along with other cells, can also be activated via the aryl hydrocarbon receptor following stimulation with Malassezia furfur-derived indole alkaloids such as malassezin, indirubin and indolocarbazole. These tryptophan-derived metabolites can trigger a variety of effects such as apoptosis of human melanocytes, but their precise role in cutaneous pathology remains to be determined. Once activated, keratinocytes can alter their cytokine expression, with up regulation of the immunosuppressive cytokines IL-10 and TGF-b and down regulation of the inflammatory cytokine IL-1a. Contradictory results have been reported for IL- 6 and TNF-a with some studies demonstrating up regulation and others showing down egulation.Keratinocytes activated by Malassezia antigens also produce antimicrobial peptides but it is not known to what extent these are protective.

角质形成细胞通过Toll样受体识别马拉色菌抗原，尽管它们与其他细胞一起也可通过芳香烃受体活化，并被糠秕马拉色菌的吲哚生物碱（例如马拉色菌素，靛玉红和吲哚咔唑）刺激激活。色氨酸衍生代谢物可以引发多种作用，例如人黑色素细胞的凋亡，但其在皮肤病理学中的确切作用有待确定。一旦激活，角质形成细胞可以改变其细胞因子的表达，同时上调免疫抑制细胞因子IL-10和TGF-b，并下调炎性细胞因子IL-1a。有与IL-6和TNF-a结果相反的报道出现，一些研究表明上调，另一些则表明下调。被马拉色菌抗原激活的角质形成细胞也产生抗菌肽，但尚不清楚其保护程度。

6.2 Activation of antigen presenting cells

6.2抗原递呈细胞的激活

Activation of antigen presenting cells has also been demonstrated in vitro using human-derived immature monocyte-derived dendritic cells (MDDCs), which are analogous to Langerhans’cells in the skin. These cells can take up whole M. furfur yeast cells and extracts, as well as recombinant M. furfur allergen 5 (Mal f 5) Mala f 1184 and M. furfur mannan. The internalisation was shown to occur via binding to the mannose receptor and pinocytosis. Subsequently, the MDDCs underwent maturation, indicated by up regulation of CD83 expression and increase in expression of the costimulatory molecules CD80 and CD86. These mature dendritic cells are excellent antigen presenting cells and are capable of presenting peptides on MHC molecules to T cells.Dendritic cells have also been shown to be activated by interaction between Malassezia antigens and various members of the C type lectin class of receptors such as Mincle, Dectins 1 & 2, and Langerin.This results in the production of pro-inflammatory cytokines such as IL-1, IL-6, IL-8 and TNF-a.A recombinant Mala f 1 gene fragment up regulated the production of IL-6, TNF-Alpha and IL-10, but not IL-12, from human leukaemia-derived dendritic cells.Interestingly, dendritic cells that have been stimulated by Malassezia antigens appear to be resistant to lysis by Natural Killer cells, a mechanism that likely favours survival of the cells in order to maintain antigen presentation.

体外使用人源性未成熟单核细胞源性树突状细胞（MDDC）证明了抗原递呈细胞的激活，该树突状细胞类似于皮肤中的郎格罕细胞。这些细胞可以吸收整个糠秕马拉色酵母菌细胞和提取物，以及重组糠秕马拉色菌过敏原5（Mal f 5）Mala f 1和糠秕马拉色菌甘露聚糖。内化作用是通过与甘露糖受体结合和胞饮作用发生的。随后，MDDCs经历了成熟，由上调CD83的表达和增强共刺激分子CD80和CD86的表达来表明。这些成熟的树突状细胞是极好的抗原递呈细胞，能将MHC分子上的肽递呈给T细胞。树突状细胞还被证明可被马拉色菌抗原与不同类型的C型凝集素类受体（例如巨噬细胞凝集素，树突状细胞凝集素1&2，郎格罕细胞凝集素）相互作用而被激活。这会产生促炎细胞因子，例如IL-1、IL-6、IL-8和TNF-α。一个重组Mala f 1基因片段上调了人白血病来源的树突状细胞的IL-6、TNF-α和IL-10的产生，但没有上调IL-12的产生。有趣的是，被马拉色菌抗原刺激的树突状细胞似乎对天然杀伤细胞的裂解有抗性，可能是一种为了维持抗原递呈有利于细胞存活的机制。

6.3 T-lymphocyte mediated immune responses

6.3T淋巴细胞介导的免疫反应

T cell-mediated immunity is important in the prevention and recovery from fungal infections and healthy human individuals demonstrate cell mediated immune responses throughout life.A deficiency in cell-mediated responses could therefore predispose the host to overgrowth of Malassezia organisms. Cell-mediated immune responses to Malassezia organisms have been investigated both in humans and dogs using assays such as leucocyte migration inhibition, peripheral blood mononuclear cell (PBMC) proliferation, immunohistochemistry, cytokine production, skin prick tests and atopy patch tests. Malassezia antigens and extracts have been shown to stimulate proliferation of PBMCs in a dose dependent manner in both humans and dogs.Furthermore, a reduction in PBMC responses was seen in seborrhoeic basset hounds compared to healthy basset hounds, although the role of this impaired response in the pathogenesis of Malassezia overgrowth in this breed is currently unknown.

T细胞介导的免疫对于预防和从真菌感染中恢复很重要，并且健康人体在整个生命中都表现出细胞介导的免疫反应。因此，细胞介导的反应不足在于可能使宿主马拉色菌过度增殖。已经在人和犬上使用例如白细胞迁移抑制、外周血液单核细胞（PBMC）增殖、免疫组化、细胞因子生产、皮肤点刺试验和特异性斑贴试验等测定方法来研究对马拉色菌的细胞介导的免疫应答。马拉色菌抗原和提取物已显示出在人和犬中以剂量依赖方式刺激外周单核细胞增殖。此外，相比于健康巴吉度猎犬，患有脂溢性皮炎的巴吉度猎犬的外周血液单核细胞应答是降低的，但尚不清楚这种应答降低在马拉色菌过度增殖的发病机制中的作用。

Despite the possible role of T lymphocytes in protective immunity, there is evidence that these cells are involved in sensitisation of humans and dogs that become allergic to the yeasts. Basset hounds with Malassezia dermatitis usually show delayed rather than immediate intradermal test reactivity to Malassezia antigens although contact sensitization, as demonstrated by patch test reactivity and characterised histologically by infiltration with CD3+ lymphocytes and neutrophils, more closely correlates with  disease  status  in  this  breed.In  both  atopic humans and dogs, PBMC responses to Malassezia spp. are exaggerated. Malassezia antigens triggered significantly higher PBMC responses in atopic people compared to healthy individuals and this effect has also been demonstrated in atopic dogs.In P. orbiculare [syn. M. globosa] patch test-positive atopic dermatitis patients, an infiltration of CD4+ T cells is seen at the patch test site,and the expression of intercellular adhesion molecule (ICAM)-1 and human leucocyte antigen (HLA)- DR in the dermis of these patients was also up regulated. Furthermore, in lesional atopic skin, the majority of the T cell clones that were reactive for P.orbiculare showed a Th2 or Th2/Th0 like cytokine profile and atopic human patients that are sensitised to Malassezia yeasts typically show increased synthesis of the Th2-related cytokines IL- 4, IL-5, IL-10 and IL-13 by Pityrosporum [Malassezia]- stimulated PBMCs.Taken together, these findings provide compelling evidence that T lymphocytes play a pivotal role in the generation of hypersensitivity reactions to Malassezia species in genetically susceptible individuals.

尽管T淋巴细胞可能在保护性免疫中发挥作用，但证据显示这些细胞参与了酵母菌对人和犬的致敏作用。虽然马拉色菌皮炎的巴吉度猎犬对于马拉色菌抗原的皮内试验通常表现出延迟反应而不是即刻反应，但接触过敏，即斑块试验反应和CD3+淋巴细胞和中性粒细胞浸润作为组织学特征，证明与该品种的疾病状况更密切相关。在特应性皮炎人和犬上，外周血液单核细胞对马拉色菌属的反应被夸大了。与健康个体相比，马拉色菌抗原在特应性皮炎人中引发的外周血液单核细胞反应明显更高，并且这种效应也已在特应性皮炎患犬中得到了证实。球形糠秕孢子菌（同义球形马拉色菌）斑贴试验阳性的特应性皮炎病患，在斑贴试验部位可见CD4+T细胞浸润，并且这些病患的真皮中的细胞间粘附分子（ICAM）-1和人白细胞抗原（HLA）-DR的表达也会上调。此外，在特应性皮炎皮肤病变中，大多数对球形糠秕孢子菌有反应的T细胞克隆显示出Th2或Th2/Th0样细胞因子谱，而对马拉色酵母菌敏感的特应性皮炎人通常会显示出增加与Th-2相关的细胞因子IL-4、IL-5、IL-10和IL-13的合成，通过被糠秕孢子菌（马拉色菌）刺激的外周血液单核细胞。总起来说，这些发现提供了令人信服的证据，证明了T淋巴细胞在对马拉色菌属遗传易感的人群的I型超敏反应中起重要作用。

6.4 IgG, IgM and IgA responses to Malassezia yeasts

6.4 lgG，lgM和lgA对马拉色酵母菌的反应

As would be expected in a typical immune response against an infectious agent, antibodies directed against antigens from Malassezia yeasts are produced in healthy humans and dogs throughout life. IgM, IgG and IgA antibodies against Malassezia species are present in both young and elderly people, but the amount of IgG and IgM tends to tail off with age corresponding with declining numbers of commensal yeasts.The Malassezia- specific IgA concentration was found to be relatively low in all age groups. In dogs, serum titres of Malassezia- specific IgG and IgA in seborrhoeic basset hounds with high cutaneous populations of

M. pachydermatis and affected dogs of various breeds significantly exceeded those of healthy basset hounds and healthy beagle dogs.Using Western immunoblotting to detect IgG responses in dogs to extracts of M. pachydermatis, four proteins of 219, 110, 71 and 42 kDa were shown to be recognised mainly by dogs with Malassezia dermatitis as compared to healthy dogs.

正如针对感染源的典型免疫应答中所预期的那样，在健康的人和犬中终生产生针对马拉色酵母菌抗原的抗体。年轻人和老年人中都存在针对马拉色菌种的lgM、lgG和lgA抗体，但lgG和lgM的含量会随着年龄增长而逐渐减少，这与共生酵母菌数量下降相对应。发现马拉色菌特异性lgA浓度在所有年龄段中都相对较低。在犬中，脂溢性皮炎的巴吉度猎犬，皮肤上有大量的厚皮马拉色菌，这些犬及各种患病纯种犬只在马拉色菌特异性lgG和lgA的血清滴度上明显超过健康的巴吉度猎犬和健康的比格犬。使用免疫印迹法检测犬对厚皮马拉色菌的提取物的lgG应答，以健康犬相比，主要有四种蛋白质（219，110，71和42 kDa）来识别马拉色菌皮炎患犬。

In humans with atopic dermatitis, the Malassezia-specific IgG concentration in adults is no different to that found in healthy individuals.However, in young atopic patients aged between 16 and 21 years, significantly elevated Malassezia-specific IgG concentrations are found.This probably reflects increased exposure to the organisms through atopic skin and a tendency for IgG to follow IgE production. Despite this, no correlation has been found between Malassezia-specific serum IgG concentration and atopy patch test responses to the yeast in patients with atopic dermatitis.It is therefore considered that determination of Malassezia-specific IgG concentrations has little value in the diagnosis of Malassezia sensitization in atopic human patients.However, concentrations of IgG4, a subtype that is induced in Th2 responses, are correlated with IgE concentrations in atopic patients with sensitivity to Malassezia sympodialis.

患有特应性皮炎的人中，成年人的马拉色菌特异性lgG浓度与健康人无异。然而，在16到21岁的特应性皮炎年轻患者中，发现马拉色菌特异性lgG浓度显著升高。这可能反应了通过特应性皮炎皮肤对生物体的暴露增加以及lgG跟随lgE产生的趋势。尽管如此，特应性皮炎患者的马拉色菌特异性血清lgG浓度与对酵母菌特异性斑贴试验反应之间未发现相关性。因此认为马拉色菌特异性lgG浓度在特应性皮炎患者马拉色菌致敏性诊断中价值不大。然而，Th2反应诱导的亚型lgG4的浓度与对合轴马拉色菌敏感的特应性皮炎患者的lgE浓度有关。

In  atopic dogs with  or without  cytological evidence of M.pachydermatis  overgrowth,there are significantly higher serum titres of Malassezia-specific IgG than those seen in healthy dogs.However, there was no significant difference between atopic dogs with or without Malassezia overgrowth. By comparing the IgG response to M. pachydermatis antigens using western immunoblotting, a protein of 25 kDa was identified in the majority of atopic dogs with Malassezia dermatitis, but only a few atopic dogs without Malassezia overgrowth and none of the normal dogs, suggesting that this protein may have some clinical relevance in the pathogenesis of Malassezia hypersensitivity.

在有或没有厚皮马拉色菌过度增殖的细胞学证据的特应性皮炎犬中，马拉色菌特异性lgG的血清滴度明显比健康犬高。然而，特应性皮炎犬无论是否患有马拉色菌过度增殖都没有显著差异。通过使用免疫印迹法比较厚皮马拉色菌抗原的lgG反应，在大多数患有马拉色菌皮炎的特应性皮炎犬中鉴定出25kDa蛋白，但也有少数特应性皮炎犬没有马拉色菌过度增殖，而正常犬都没有这种情况，这表明蛋白可能与马拉色菌超敏反应的发病机制间的临床相关性。

In summary, it is clear that IgG responses to Malassezia yeasts are common in both healthy humans and dogs. This probably reflects exposure of the immune system to antigens produced by commensal organisms. However, enhanced IgG responses can be seen in dogs with Malassezia dermatitis and in humans and dogs with atopic dermatitis. The role of this IgG response in the pathogenesis of skin disease is currently unclear, both in humans and dogs. However, as overgrowth  with Malassezia organisms does not appear to be a self-resolving condition, it seems likely that these antibodies are not protective. Alternatively, IgG antibodies could activate the complement system, as has been demonstrated with Pityrosporum ovale [M. furfur] and P. orbiculare [M. globosa],and exacerbate the inflammatory response. A final possibility is that IgG responses to the yeast are merely an epiphenomenon and neither contribute to, nor inhibit, the ongoing disease process. Further studies are therefore required to determine the precise role played by these antibodies in Malassezia-induced skin disease.

综上所述，很明显在健康人和犬中对马拉色酵母菌的lgG反应很常见。这可能反应出免疫系统暴露于共生生物产生的抗原。然而，在患有马拉色菌皮炎的犬及特应性皮炎的人和犬中，可以看到lgG反应的增强。在人和犬中这种lgG反应在皮肤疾病的发病机理中的作用尚不清楚。然而，马拉色菌过度增殖似乎不是一种自愈性疾病，因此这些抗体似乎没有保护性。另外，lgG抗体可以激活补体系统，例如被证实的糠秕马拉色菌和球形马拉色菌一样，并会加剧炎症反应。最后一种可能性是，对酵母菌的lgG反应只是一种现象，在正在进行的疾病过程中既不促进也不抑制。因此，需要进一步的研究来确定这些抗体在马拉色菌引起的皮肤疾病中发挥的确切作用。

6.5 IgE responses to Malassezia yeasts

6.5马拉色酵母菌的lgE反应

Increased concentrations of Malassezia-specific IgE are frequently present in atopic humans and dogs. Multiple studies have shown that IgE responses to Malassezia organisms are significantly higher in human patients with atopic dermatitis compared to those with other atopic diseases or healthy individuals.In human patients with atopic dermatitis of the head and neck, the clinical severity of the condition is significantly correlated with the concentration of anti M. furfur IgE. Furthermore, IgE production by PBMCs stimulated with Malassezia extracts in vitro was significantly higher in radioallergosorbent test (RAST)+ atopic dermatitis patients compared with RAST- atopic dermatitis patients or healthy controls.This Malassezia-specific IgE has been found to correlate with the degree of response to Malassezia extracts in atopy patch tests at 48 h post-test in atopic dermatitis patients.

在特应性皮炎人和犬中经常存在马拉色菌特异性lgE浓度的升高。多项研究表明，特应性皮炎患者的lgE对马拉色菌的反应比其他特应性疾病或健康个体明显更高。在特应性皮炎患者的头部和颈部，临床症状的严重程度与抗糠秕马拉色菌lgE浓度显著相关。此外，在放射性变态吸附试验（RAST）+的特应性皮炎患者中，马拉色菌提取物刺激的外周血液单核细胞体外产生的lgE明显于RAST-的特应性皮炎患者或健康对照者。在特应性皮炎患者测试后48小时后，特应性斑贴试验中发现这种马拉色特异性lgE与马拉色菌提取物反应程度有关。

In humans, multiple proteins from Malassezia yeasts ranging in molecular size from 9–110 kDa have been characterised as major allergens in atopic dermatitis and a number have been sequenced and cloned, including Mal f 1–9 and Mal s 1–13.One particular allergen, Mala s 13, a thioredoxin enzyme, can crossreact with the human form of the enzyme, leading to the production of autoreactive T cells that can sustain ongoing skin inflammation.A similar effect has been reported for Mala s 11, a manganese-dependent superoxide dismutase enzyme that is found in both yeast and human cells.

在人中，马拉色酵母菌多种蛋白质中分子量从9-110kDa不等，已被鉴定为特应性皮炎的主要过敏原，并已测序和克隆多种蛋白质，包括Mal f1-9和Mal s 1-13.一种特殊的过敏原，Mala s 13，一种硫氧还蛋白酶，可以人体的酶发生交叉反应，导致产生可以维持皮肤炎症持续存在的自发性T细胞。据类似报道，Mala s 11是一种在酵母菌和人体细胞中都发现的锰依赖性超氧化物歧化酶。

In dogs, concentrations of IgE antibodies to M. pachydermatis are significantly higher in atopic dogs than in healthy dogs or non-atopic dogs with Malassezia overgrowth.However, dogs with recurrent Malassezia otitis had similar concentrations of allergen-specific IgE to those with healthy ears, suggesting that hypersensitivity is not always involved in such infections.Using Western immunoblotting to characterise individual antigen responses, proteins with molecular weights of 45, 52, 56 and 63 kDa from M. pachydermatis have been demonstrated to be major allergens in atopic dogs with Malassezia overgrowth.

在犬中，特应性皮炎患犬中针对针对厚皮马拉色菌的lgE抗体的浓度显著高于健康犬或马拉色菌过度增殖的非特应性皮炎患犬。然而，复发性马拉色菌性耳炎的犬的过敏原特异性lgE浓度与正常耳道的相似，暗示着过敏不总是参与这类感染。使用免疫印迹法来表征单个抗原反应，已证明来自厚皮马拉色菌的分子量为45，52，56和63kDa的蛋白质是患有马拉色菌过度增殖的特应性皮炎患犬的主要的过敏原。

These studies provide convincing evidence that proteins from Malassezia yeasts can act as allergens in dogs predisposed to the development of atopic dermatitis.

这些研究提供了令人信服的证据，表明马拉色酵母菌中的蛋白质可以在易患有特应性皮炎的犬中充当过敏原。

6.6 Mast cell responses

6.6肥大细胞反应

Studies have indicated that mast cells may be involved in innate immune responses against Malassezia spp. The interaction between Curdlan, a ß-glucan component of the cell wall of M. sympodialis, and C-type lectin pattern recognition receptors (such as Dectin-1) expressed on the surface of human cutaneous mast cells, induced degranulation and augmented IgE-mediated granule exocytosis.In other circumstances, fungal ligands may induce Dectin-1 mediated mast cell activation and production of chemokines and cytokines without degranulation.

研究表明，肥大细胞可能参与了针对马拉色菌的先天免疫反应。凝胶多糖，合轴马拉色菌细胞壁的ß-葡聚糖成分与人皮肤肥大细胞表面表达的C型凝集素模式识别受体（如树突状细胞凝集素-1）三者之间的相互作用可引起细胞脱颗粒，并增加lgE介导的颗粒胞吐作用。在其他情况下，真菌配体可能诱导树突状细胞凝集素-1介导的肥大细胞活化以及产生趋化因子和细胞因子，而无脱颗粒作用。

In order to demonstrate full IgE-mediated hypersensitivity, it is necessary to document mast cell degranulation following allergen exposure. In humans, both intradermal tests (IDT) and skin prick tests (SPT) may show positive reactivity to Malassezia allergens in patients suffering from atopic diseases,whereas these tests are usually negative in healthy controls. Stronger reactions are typically seen in patients with generalised atopic dermatitis,or those with lesions predominantly on the head and neck.The SPT results have also been found to correlate with levels of Malassezia-specific IgE in the serum and with results of basophil histamine release tests but not with the severity of atopic dermatitis.

为了证明完全由lgE介导的超敏反应，有必要记录过敏原暴露后肥大细胞脱颗粒。在人中，皮内试验（IDT）和皮肤点刺试验（SPT）均可显示患有特应性疾病的患者对马拉色菌过敏原的阳性反应，而这些试验的健康对照中通常都是阴性的。一般特应性皮炎患者或者主要病灶在头颈部的患者通常会出现更强的反应。SPT结果以及嗜碱性粒细胞组织胺释放试验结果还与血清中马拉色菌特异性lgE水平相关，但与特应性皮炎的严重程度无关。

Positive IDT results to Malassezia extracts have also been reported in atopic dogs.Immediate hypersensitivity responses to intradermal injections of M. pachydermatis extracts at concentrations which caused no reaction in healthy dogs have been observed in atopic dogs with Malassezia dermatitis, although they were also seen in some atopic dogs without Malassezia dermatitis.Nevertheless, the reactivity to the extracts in atopic dogs with cytological evidence of Malassezia overgrowth was significantly higher than that in atopic dogs without. Additionally, positive immediate hypersensitivity reactions to extracts from M.pachydermatis using Prausnitz-KÜstner tests have been demonstrated.Clinically normal dogs received pooled sera from atopic dogs with Malassezia dermatitis that were IDT positive to Malassezia extracts and serum from an atopic dog with Malassezia dermatitis exhibiting high levels of anti-Malassezia IgE on an ELISA assay. Positive IDT responses were observed in the recipients following subsequent injection of the yeast extract, indicating that anti-Malassezia IgE antibodies are functional in Type I hypersensitivity reactions.As with intradermal testing to other allergens, there is reasonable agreement between the results of intradermal tests and IgE serology to yeast allergens, although it is possible to record a positive result in one test and a negative result in the other.The frequency of immediate hypersensitivity responses to M.pachydermatis extracts in non-atopic dogs with Malassezia dermatitis is low. One study investigating IDT reactivity to M. pachydermatis in eight healthy basset hounds, 17 basset hounds with Malassezia dermatitis and 19 healthy beagle dogs, reported that only two affected basset hounds and one healthy beagle dog showed immediate hypersensitivity reactions.Taken together, these findings suggest that mast cell mediated hypersensitivity responses to M. pachydermatis allergens may be involved in the pathogenesis and contribute to the clinical signs in many cases of cAD. As a result of this Malassezia allergens are now typically included in standard intradermal allergen tests and IgE serology, although, to date, there is relatively limited evidence of beneficial effects of allergen-specific immunotherapy against M. pachydermatis in dogs.

在特应性皮炎患犬中也报道了对马拉色菌提取物的IDT的阳性结果。在患有马拉色菌皮炎的特应性皮炎患犬中，皮内注射厚皮马拉色菌提取物立刻引起超敏反应，但对健康犬无反应，在没有马拉色菌皮炎的特应性皮炎患犬中也有观察到这一现象。尽管如此，具有细胞学证据的马拉色菌过度增殖的特应性皮炎患犬对提取物的反应明显高于没有马拉色菌过度增殖的特应性皮炎患犬。此外，已证明使用Prausnitz-Kustner试验厚皮马拉色菌提取物会立刻引起超敏反应。临床上健康犬得到了从患有马拉色菌皮炎的特应性皮炎患犬上提取的混合血清，这些血清对马拉色菌提取物呈IDT阳性，并且患有马拉色菌皮炎的特应性皮炎患犬的血清在酶联免疫吸附试验中展示了很高水平的抗马拉色菌lgE。随后注射酵母菌提取物后，在受体中观察到IDT阳性反应，表明抗马拉色菌lgE抗体在I型超敏反应中起作用。与皮内测试其他过敏原一样，皮内测试结果与针对酵母过敏原的lgE血清学检测之间存在合理性的一致，尽管有可能在一次测试中记录了阳性结果而在另一次测试中记录了阴性结果。在患有马拉色菌皮炎的非特应性皮炎患犬中，对厚皮马拉色菌提取物立刻显示超敏反应的频率很低。一项关于IDT对厚皮马拉色菌的反应的研究，在八只健康的巴吉度猎犬，17只患有马拉色菌皮炎的巴吉度猎犬和19只健康的比格犬中，只有两只患病巴吉度猎犬和一只健康的比格犬表现出立刻的超敏反应。综合起来，这些发现表明肥大细胞介导的对厚皮马拉色菌过敏原的超敏反应引起了疾病，并在许多cAD病例中产生了临床症状。因此这种马拉色菌过敏原是现在非常典型的，包括了标准的皮内过敏原测试和lgE血清学，尽管迄今为止，相对有限的证据显示，过敏原特异性免疫疗法对抗犬的厚皮马拉色菌有效。

6.7Conclusions

6.7结论

Based on the above studies, the likely sequence of events leading to immune responses to Malassezia yeasts can be summarized as follows:

基于以上研究，可能引起马拉色酵母菌免疫应答的一系列事件总结如下：

1.Malassezia species proliferating within the stratum corneum of dogs (and humans) produce numerous antigens and allergens.

1.马拉色菌在犬（和人）的角质层中增殖，产生大量抗原和过敏原。

2.These antigens penetrate to the living epidermis and activate keratinocytes, resulting in the production of antimicrobial peptides and alterations in cytokine expression.

2.这些抗原渗透到活表皮层并激活角质形成细胞，从而导致抗菌肽的产生和细胞因子表达的改变。

3.Malassezia-derived antigens that permeate into and through the living epidermis are captured by epidermal Langerhans’ cells and/or dermal dendritic antigen-presenting cells.

3.马拉色菌来源的抗原渗透并穿过活表皮层，被表皮的郎格罕细胞和/或真皮树突状抗原递呈细胞捕获。

4.These cells then migrate to regional lymph nodes and present the antigen to a T lymphocyte via a major histocompatibility complex (MHC) class II molecule.

4.然后这些细胞迁移到淋巴结区域，并通过主要组织相容性复合体（MHC）II类分子将抗原递呈给T淋巴细胞。

5.In cooperation with different cytokines, T helper (Th) 0 precursor cells differentiate into Th1 cells and/or Th2 cells. A cytokine environment dominated by IL-12 would favour Th1 cell development, whereas IL- 4 and IL-13 would stimulate the development of Th2 cells.

5.与不同的细胞因子协同合作，T辅助（Th）0前体细胞分化为Th1细胞和/或Th2细胞。IL-12为主的细胞因子将有利于Th1细胞的发育，而IL-4和IL-13将刺激Th2细胞的发育。

6.T helper cells would activate B lymphocytes and stimulate them to differentiate into antibody–forming plasma cells. By secreting IL-2 and IFN-c, Th1 cells would promote IgG production, whereas IL-4 and IL-13 from Th2 cells would promote immunoglobulin class switching to IgE.

6.T辅助细胞会激活B淋巴细胞并刺激它们分化为产生抗体的浆细胞。Th1细胞通过分泌IL-2和IFN-c，促进lgG的产生，而Th2细胞分泌IL-4和IL-13将促进免疫球蛋白类别转换成lgE。

7.The production of Malassezia-specific IgG antibodies could potentially provide a degree of protective immunity against Malassezia organisms. Alternatively, these antibodies might activate the complement system causing epidermal damage and inflammation.

7.马拉色菌特异性lgG抗体的产生可能潜在地提供针对马拉色菌生物体一定程度上的保护性免疫。或者，这些抗体可能激活补体系统，导致表皮损伤和炎症。

8.The development of allergen-specific IgE antibodies could lead to sensitization of cutaneous mast cells. Subsequent exposure to Malassezia allergens could trigger the release of inflammatory mediators, resulting in a Type I hypersensitivity reaction.

8.过敏原特异性lgE抗体的发展可能导致皮肤肥大细胞致敏。随后暴露于马拉色菌过敏原可能触发炎症介质的释放，导致I型超敏反应。

9.Circulating populations of Malassezia-specific sensitised T-cells might mediate delayed and contact hypersensitivity in selected patients.

9.循环中的马拉色菌特异性敏化T细胞群体可能会介导某些患者的迟发性和接触性超敏反应。

6.8Implications for clinical diagnosis and therapy

6.8对临床诊断和治疗的意义

1.A range of immunological hyper-responsiveness can be present in dogs with  Malassezia dermatitis (none, immediate, delayed, contact). Tests for immediate hypersensitivity (serology, intradermal) are relatively accessible (although not standardised) whereas delayed reactivity following intradermal testing is assessed infrequently and patch testing is technically challenging in the clinical environment.

1.马拉色菌皮炎患犬可能存在多种免疫性超敏反应（无、速发、迟发、接触）。速发超敏反应（血清学、皮内试验）相对容易（但尚未标准化），而皮内测试后的迟发反应很少被评估，贴片试验在临床环境中在技术上具有挑战性。

2.Serological and skin test reactivity is also seen in a proportion of unaffected dogs; thus immunological tests must be assessed in the context of clinical and cytological data; they should not be used as standalone ‘diagnostic’ tests.

2.血清学和皮肤测试反应性也见于部分未患病犬中；因此免疫学检测必须根据临床和细胞学检查进行评估；它们不应被应用作为独立的诊断测试。

3.It is intuitive that evidence of immediate, IgE-mediated or cellular hypersensitivity might indicate the need for rigorous antifungal therapy to minimize allergen challenge in the sensitized host, although this remains to be proven by controlled therapeutic studies.

3.简言之，能证明的速发的、lgE介导的或细胞超敏反应，可能显示出需要严格的抗真菌治疗，以最大程度减少致敏宿主中的过敏原攻击，但这仍然需要由对照组治疗研究证明。

4.Although evidence of immediate, IgE-mediated hypersensitivity provides a rationale for allergen-specific immunotherapy (AIT), to date, there is relatively limited evidence of beneficial effects of AIT against M. pachydermatis in dogs. More data is required before this can be systematically recommended.

4.虽然，速发的、lgE介导的超敏反应为过敏原特异性免疫治疗（AIT）提供了理论依据，但迄今为止，AIT对厚皮马拉色菌患犬有效的证据相对有限。系统地介绍这些之前还需要更多的数据支持。

5.Whilst some laboratories offer serological testing for IgG reactivity to M. pachydermatis, the clinical utility of this test is uncertain because there is no evidence of any diagnostic or therapeutic value.

5.虽然一些实验室提供针对厚皮马拉色菌的lgG反应的血清学检测，但该检测的临床效用不准确，因为没有诊断或治疗价值的证据。

7.Predisposing factors for development ofMalassezia dermatitis

7.马拉色菌皮炎发展的易感因素

Overgrowth of commensal Malassezia yeasts may occur due to alterations of the skin’s surface microclimate, leading to inflammatory skin disease.Primary diseases that cause increased moisture, altered surface lipids, and/or disruption of stratum corneum barrier function, or aberrant immune responses may encourage this secondary process of overgrowth.

由于皮肤表面微气候的变化，共生的马拉色酵母菌可能会过度增殖，导致炎性皮肤病。原发疾病会导致水分增加，表面脂质改变和/或角质层屏障功能受损，或异常的免疫反应可能会促进继发过度增殖。

7.1 The role of gender, age and breed

7.1性别、年龄和品种的作用

Gender and age do not appear to be consistently correlated with the presence of Malassezia dermatitis, but breed predilections in dogs have been described in several studies where cases were compared to the institutions’general hospita or diagnostic laboratory populations. Breeds identified to be at increased risk by these studies include West Highland white terriers (WHWT), English setters, shih tzus, basset hounds,American cocker spaniels, boxers, dachshunds, poodles and Australian silky terriers. Breeds with conformations that favour skin folds are also prone to infections at intertriginous anatomical sites. The basset hound and WHWT in particular demonstrate clinically distinctive conditions characterized by generalized seborrhoea (basset hound)or generalized, severely pruritic dermatitis with marked lichenification and hyperpigmentation.It is noteworthy that all of these breeds are recognized to be at increased risk for developing ether atopic dermatitis or primary idiopathic seborrhoea. However, basset hounds in the UK that present with (an albeit relapsing) M. pachydermatis-associated seborrhoeic presentation typically respond dramatically to antimicrobial shampoo therapy with no residual clinical signs of atopic or seborrhoeic dermatitis.

性别和年龄似乎与马拉色菌皮炎的存在并不总是相关的，但是在几项研究中描述了犬品种的倾向性，其中将病例与机构总医院或诊断实验室的群体进行了比较。这些研究确定了高风险的品种包括了西高地白㹴（WHWT）、英国雪达犬、西施犬、巴吉度猎犬、美国可卡犬、拳狮犬、腊肠犬、贵宾犬和澳大利亚丝毛㹴。具有皮肤皱褶构造的品种犬也容易在摩擦部位感染。尤其是巴吉度猎犬和西高地白㹴会表现出特别的临床症状，特征为全身性脂溢性皮炎（巴吉度猎犬）或者全身性严重的瘙痒性皮炎，具有明显的苔藓化和色素沉着。值得注意的是，所有这些品种都被认为患特应性皮炎或原发性脂溢性皮炎的风险较高。然而在英国出现了与厚皮马拉色菌相关的脂溢性皮炎（尽管是复发性的）的巴吉度猎犬，对抗菌药浴香波治疗显著有效，没有特应性皮炎或脂溢性皮炎的其余症状。

Likewise, predisposed feline breeds have been recognized, although not through statistical analyses as compared to general hospital populations. Case-control studies in Devon rex cats and the sphynx breed, have demonstrated increased Malassezia counts as compared to domestic shorthaired cats.In these cases, Malassezia overgrowth is associated  with the clinical finding of seborrhoeic skin; especially in the claw folds.

同样，虽然没有通过统计分析与一般医院群体进行比较，但已认识到易患的猫科品种。在病例对照研究中，德文卷毛猫和斯芬克斯猫中马拉色菌数量高于家养短毛猫。在这些病例中，马拉色菌过度增殖与临床发现的脂溢性皮炎相关，尤其是爪部褶皱处。

7.2 The role of cutaneous hypersensitivity disorders in the host

7.2皮肤过敏疾病在宿主中的作用

Pruritic inflammatory diseases such as atopic dermatitis may create microclimate changes due to scratching (disruption of barrier function), licking (added moisture) or increased production of sebum.Despite the clinical observation that Malassezia overgrowth and atopic dermatitis commonly cooccur,studies have sometimes failed to demonstrate a clear statistical association between the two conditions.Nevertheless it is generally accepted that M. pachydermatis may increase the inflammation associated with cAD directly by induction of inflammatory cytokines from epidermal cells and indirectly by acting as an allergen(Section 6). Thus, a hypersensitivity response to M.pachydermatis might explain the discordance in the studies mentioned above since density of yeast on the skin’s surface cannot be used as the sole determinant of its pathogenic effect.However, in a study where cAD was the predominant diagnosis for a group of dogs with inflammatory skin lesions, there was a statistically significant correlation between higher CADESI-03 scores and increased colony forming units of M.pachydermatis isolated by quantitative culture.The range of epidermal barrier defects that might influence Malassezia-host interactions in cAD has been the subject of a detailed review. Malassezia spp. overgrowth has also been reported in series of cats with allergic skin disease.

瘙痒性炎性疾病，如特应性皮炎，可能会由于抓挠（破坏屏障功能），舔舐（增加水分）或者增加皮脂产生，可能会造成微环境改变。虽然临床观察到马拉色菌过度增殖和特应性皮炎通常是同时发生的，但研究有时未能证明这两者之间明确统计学联系。不过一般来说厚皮马拉色菌直接与cAD联合，可能会使炎症增加，通过诱导来自表皮细胞的炎性细胞因子，并且直接充当过敏原。因此，过敏症对厚皮马拉色菌的反应性可能是上述研究保持了谨慎，因为无法将酵母菌在皮肤表面的密度作为其致病作用的唯一决定因素。然而，在一项研究中，以cAD作为患有炎性皮肤病变犬的主要诊断，得到了高分CADESI-03和定量培养分离出的厚皮马拉色菌菌落形成单位之间的统计学上的显著相关性。在cAD中可能影响马拉色菌宿主的表皮屏障缺陷已成为一个需详细探讨的课题。马拉色菌过度增殖在一些猫过敏性皮肤病上也有报道。

7.3The role of seborrhoeic dermatoses and aberrant cornification

7.3脂溢性皮肤病和角化异常的作用

Primary and secondary seborrhoeic conditions also favour proliferation of Malassezia spp. Seborrhoeic dermatoses in dogs have been statistically associated with significantly  higher M.pachydermatis skin surface counts as compared to healthy dogs.Similar associations have been described in cats with seborrhoeic disorders.Endocrinopathies such as hypothyroidism and hyperadrenocorticism may cause secondary seborrhoea in dogs;while no studies have demonstrated either disease to be an independent risk factor for Malassezia overgrowth, cases where an endocrinopathy and Malassezia dermatitis have occurred concurrently have been reported.In cats, no differences in Malassezia carriage were noted between healthy controls and cases with either hyperthyroidism or diabetes mellitus, using a contact plate technique for quantitative culture at several body sites; however, it should be noted that the cats with endocrinopathies in this study had no skin lesions at the time of sampling.

原发性和继发性脂溢性疾病也有利于马拉色菌的增殖。据统计，与健康犬相比，犬脂溢性皮炎与厚皮马拉色菌的皮肤表面高数量计数显著相关。在脂溢性皮炎的猫中也有类似的关联。在犬中内分泌疾病如甲减和肾亢可能导致继发脂溢性皮炎；虽然尚无研究表明这两种疾病是导致马拉色菌过度增殖的一个独立危险因素，但已报道了同时发生内分泌病和马拉色菌皮炎的病例。在猫中，健康对照组和患有甲亢或糖尿病的猫之间没有发现马拉色菌携带的差异，使用了接触板技术在多个身体部位进行定量培养；然而，应该注意的是在这项研究中，患有内分泌病的猫在采样的时候没有皮肤病变。

Malassezia overgrowth appears to be favoured by skin diseases with (assumedly) altered stratum corneum barrier function through aberrant cornification. Diseases of dogs characterized histopathologicaly by parakeratotic hyperkeratosis-such as zinc-responsive dermatosis and hepatocutaneous syndrome/superficial necrolytic dermatitis-have been anecdotally reported to promote yeast overgrowth (Section 10).The presence of Malassezia overgrowth is associated with pruritus in American bulldogs with autosomal recessive congenital ichthyosis caused by a single base deletion in the gene NIPAL4.In cats, a histological study of 550 skin biopsy cases identified Malassezia overgrowth most commonly with thymoma-associated dermatosis (TAD) and paraneoplastic alopecia (PNA) associated with internal neoplasia.In TAD, parakeratosis is a common histopathological feature, while PNA presents either with absence of a stratum corneum or some degree of parakeratosis.In one cat with TAD, resolution of Malassezia dermatitis was reported to occur after its thymoma was surgically excised.It is important to recognize that these diseases are not typically pruritic unless Malassezia overgrowth is present. Feline facial acne, thought to represent an idiopathic disorder of follicular keratinization, may also result in Malassezia overgrowth.

马拉色菌的过度增殖似乎受到皮肤疾病的影响，皮肤疾病通过角化异常（据认为）改变了角质层的屏障功能。犬中以角化过度为特征的角化病，例如锌反应性皮炎和肝皮综合症/浅表性坏死性皮炎，在组织病理学上会促进酵母菌过度增殖。马拉色菌过度增殖的存在与美国斗牛犬的在NIPAL4基因中缺失一个碱基导致的常染色体隐性遗传的先天性鱼鳞病引起的瘙痒症有关。在猫中，对550例皮肤活检病例的组织学研究发现，马拉色菌过度增殖最常见于胸腺瘤相关性皮肤病（TAD）和副肿瘤性脱毛（PNA）。在TAD中，角化不全是常见的组织病理学特征，而PNA则表现为无角质层或某种程度的角化不全。据报道，在一只患有TAD的猫中，马拉色菌性皮炎消退是手术切除胸腺瘤后发生的。重要的是认识到除非出现马拉色菌过度增殖，否则这些疾病通常不会瘙痒。猫面部痤疮被认为是特发性的毛囊角质化，也可能导致马拉色菌过度增殖。

7.4The role of climate

7.4气候的作用

The skin surface presents a range of natural microclimates and several ecological niches with different moisture and nutrient levels may be recognised.The eyes, ears, nares, oral cavity, lip fold, prepuce, vagina and anus provide microenvironments that are moist with secretions and constitute unique ecological niches (Section 4). The skin also presents gross topographical features that affect moisture and retain secretions such as the intertriginous zones and the interdigital spaces. It is from these areas that Malassezia overgrowth commonly develops. The external (macro-) environment may also play a role in predisposing dogs to Malassezia overgrowth. It is generally recognised that Malassezia dermatitis is more common in tropical climates and during warm, humid months in more temperate latitudes, in accordance with the concept that Malassezia yeasts inhabit a‘transitional mantel zone’that is influenced by both host skin and the animal’s external environment.Although not specifically studied in the dog, this factor is well-documented in human medicine.

皮肤表面呈现出一系列自然的微气候，并且可以识别出具有不同水分和营养水平的几个生态位。眼部、耳部、鼻部、口腔、唇褶、包皮过长、阴道和肛门提供了微环境，湿润的分泌物构成了独特的生态位。皮肤还呈现出影响水分和保留分泌物的特性，例如间擦区和指间缝隙。这些区域会促使马拉色菌过度增殖。外部（宏观）环境也可能使犬容易出现马拉色菌过度增殖。人们普遍认为，马拉色菌皮炎在热带气候中以及在温带气候地区更温暖潮湿的月份更容易生长，这是根据马拉色酵母菌居住在一个受宿主皮肤和动物的外部环境影响而形成的一个过度区域的概念得出的。尽管在犬中没有专门研究，这一因素已在人类医学中得到充分证明。

7.5Factors of uncertain importance

7.5不确定的重要因素

The role of immunosuppression as a risk factor for Malassezia overgrowth has been discussed anecdotally,but no studies have examined the immunosuppressed state as a risk factor for animals with active yeast infection. In cats without skin disease, retroviral infection was associ- ated with increased density of Malassezia spp. isolation from the hair coat as compared to non-infected cats.

免疫抑制作为马拉色菌过度增殖的危险因素已有传闻，但尚无研究免疫抑制状态是活跃酵母菌感染的危险因素。在没有皮肤病的猫中，与没有感染的猫对比，逆转录病毒感染与从毛发分离的马拉色菌的密度增长有关。

Antibacterial therapy has not been reported to be an independent risk factor for development of Malassezia dermatitis in any published studies. However, a correlation between the onset of Malassezia dermatitis (or otitis) and the recent use of antibacterial drugs is sometimes observed by practitioners and could reflect a reduction in“competition”for micro-ecological resources as the bacterial population is reduced. However, the opposite has also been noted for individual dogs, where yeast counts dropped following treatment with cefalexin.

在已发表的研究中，尚未报道抗菌疗法是导致马拉色菌皮炎发展的独立危险因素。但是，从业人员有时会观察到马拉色菌性皮炎（或耳炎）的发作与近期使用抗菌药物之间的相关性，这可能反映出随着细菌数量的减少，微生态资源的竞争减少了。但是，对于个别犬也存在相反的情况，其中头孢氨苄治疗后酵母菌数量下降。

7.6‘Idiopathic’ cases

7.6特发性病例

In a proportion of dogs and cats with Malassezia dermatitis, no concurrent disease or predisposing factors are identified.This is analogous to “idiopathic recurrent pyoderma”and likely reflects incomplete understanding of, and an inability to investigate and define in detail any impairment of, the complex coordinated innate and adaptive immune events that determine the outcome of skin colonisation by these yeasts (Section 6).

在一部分患有马拉色菌皮炎的犬和猫中，未发现并发疾病或诱发因素。这类似于特发性复发性脓皮病，并且可能反映出理解的不完整性，无法详细研究和定义任何损伤复杂的协调先天性和适用性免疫事件，这些事件决定了这些酵母菌在皮肤上定植的结果（第6节）。

7.7Conclusions

7.7结论

Dog breeds identified to be at increased risk of Malassezia dermatitis include West Highland white terriers (WHWT), English setters, shih tzus, basset hounds, American cocker spaniels, boxers, dachshunds, poodles and Australian silky terriers. Devon rex and sphynx cats are also predisposed. The presence of skin folds is a common risk-factor for localised disease. Dogs with Malassezia dermatitis often have concurrent hypersensitivity disorders, cornification defects or endocrinopathies. Cats without a breed predilection most often have an underlying hypersensitivity disorder, visceral neoplasia or other serious internal disease.

被确定患有马拉色菌皮炎风险较高的犬种包括西高地白㹴（WHWT）、英国雪达犬、西施犬、巴吉度猎犬、美国可卡犬、拳狮犬、腊肠犬、贵宾犬和澳大利亚丝毛㹴。德文卷毛猫和斯芬克斯猫也很容易出现。皮肤褶皱的存在是局部疾病常见的危险因素。患有马拉色菌皮炎的犬常会出现并发的过敏性疾病、角化缺损或内分泌疾病。没有品种易感性的猫，通常会患有潜在的过敏症、内脏肿瘤或其他严重的内部疾病。

8.Quantification of Malassezia populations on canine and feline skin by cytology and culture

8.通过细胞学检查和培养，对犬猫皮肤上马拉色菌种群进行定量

8.1Background

8.1背景

The assessment of the presence and number of Malassezia species is an important step in the characterisation of the cutaneous ecosystem of dogs and cats in both health and disease. Studies utilising traditional cytological and cultural methods have clearly demonstrated that Malassezia yeasts are normal inhabitants of healthy canine and feline skin and mucosae (Section 4).It is also clear that Malassezia overgrowth is a feature of a variety of clinical presentations in dogs and cats.More recently developed molecular techniques have the advantage of being able to detect minority populations or organisms that cannot be cultivated using routine methods,an issue that hindered progress with the lipid-dependent Malassezia spp. for decades.The molecular microbiome approach has led to a marked expansion of the diversity of bacterial genera shown to inhabit skin.The Malassezia genus has retained its status as the dominant fungal inhabitant of this ecological niche in humans, whereas dog and cat studies have shown a wider diversity of fungal genera, normally associated with an environmental source (Section 4). The relatively low abundance of Malassezia spp. likely reflects the detection of transient environmental visitors rather than permanent residents.

对马拉色菌属物种的存在和数量的评估是在健康和疾病方面表征犬和猫皮肤生态系统的重要步骤。利用传统细胞学和培养的方法进行的研究清楚地表明，健康犬和猫皮肤和粘膜是马拉色酵母菌的正常栖息地。显然，马拉色菌过度增殖在犬猫中有各种临床症状。最近开发的分子技术的优势是能够检测无法使用常规方法培养的少数群体或生物，该问题阻碍了脂质依赖性马拉色菌的发展。分子微生物方法导致居住在皮肤上的细菌多样性扩大。马拉色菌属一直是人生态位的主要真菌栖息地，而犬猫的研究表明，真菌属多样性更大，通常与环境有关。马拉色菌属的相对较低的丰度可能反应了对短暂环境访问者而非永久居民的检测。

Researchers and clinicians have developed a range of semi-quantitative and quantitative methods for enumeration of yeasts in skin, some of which have important applications in veterinary clinical practice for routine diagnosis, assessment of response to therapy, and in research and development of novel therapeutic agents and formulations. However, it is important to recognise that each have limitations that affect the final count;methods suitable for rapid assessment of Malassezia populations by veterinary practitioners‘on the clinic floor’are not necessarily optimal for scientific research. The relative lack of sensitivity of histopathology for detecting Malassezia spp. in the stratum corneum is discussed in Section 10.

研究人员和临床医生开发了一系列半定量和定量的方法用于皮肤中酵母菌的计数，其中一些方法在兽医临床实践中用于常规诊断，对治疗的反应评估以及在新型治疗剂的研发中具有重要的应用和规划。然而，重要的认识到每种方法都有影响到最终计数的局限性，适用于快速评估马拉色菌种群的方法，兽医们在诊所上不一定适合科学研究。组织病理学检测马拉色菌的相对敏感型，第10节将讨论角质层中的马拉色菌属。

8.2Development and utility of skin sampling techniques for quantification of cutaneous Malassezia populations

8.2皮肤采样技术在皮肤马拉色菌中定量研究的开发和应用

Methods for microbiological assessment of skin populations have traditionally included impression (cytology using slides or tape; culture) and dispersal (primarily cupscrub or swabwash) methods.Impression culture methods tend to underestimate microbial populations whereas dispersal methods yield values closer to the true population. Cytological examination without impression (scrapings, swab samples) are more recent developments.

皮肤种群的微生物学评估方法传统上包括皮肤压片法（使用载玻片或胶带进行细胞学；培养）和分散法（主要是杯刷法或拭子清洗法）。压片培养法往往会低估微生物菌群，而分散法会产生微生物值。无压片的细胞学检查（皮肤刮片、拭子采样）是最近的发展。

8.2.1 Impression cytology and related techniques

8.2.1压片细胞学及相关技术

Development. Early studies primarily utilised cytological techniques as Malassezia dermatitis emerged as an important inflammatory disease of dogs. These included scraping methods,direct impression with glass slides,or slide preparations prepared by rolling swabs previously rubbed on the lesional skin.One study elegantly described the utility of the direct application of vinyl adhesive tape in the assessment of cutaneous bacterial and fungal populations in human skin.A comparison of effectiveness of staining methods for detecting Malassezia cells in specimens from animals seems lacking, perhaps reflecting widespread satisfaction with a modified Wright-Giemsa stain (“Diff-Quik” [Harleco, NJ, USA] or generic equivalent) (Figure 3) that is likely to be available in the clinical area,67,266,267 although cotton-blue lactopheno and May-Grunwald Giemsa stains have also been used.

发展。马拉色菌性皮炎是犬一种重要的炎性疾病，早期研究主要利用细胞学技术。这些包括皮肤刮片法、载玻片直接压片法，或皮肤病变采样拭子在载玻片滚动制片。一项研究描述了直接将乙烯基胶带粘贴皮肤的用途。似乎缺乏对动物样本中马拉色菌细胞进行检测的染色方法有效性的比较，这可能反映出人对改良版瑞氏-姬姆染色（Diff-Quik）会在临床使用，但也使用了棉蓝乳酚和梅吉染色法。

Utility. Tape-stripping has gained wide acceptance inveterinary clinical practice as a rapid and versatile method for recovering stratum corneum cells and their attendant adherent microbes.It has also been used in a number of therapeutic studies,although these publications should be interpreted with due regard to the limitations of this method discussed below. Adhesive tape can be applied to deeply folded or recessed areas that are not readily accessible for direct slide application, often with minimal animal restraint. The adhesive properties aid removal of skin surface material especially in erythematous lesions with limited exudation. By counting yeast cells in a certain number of microscopical fields in the tape-strip sample, a known area of skin surface is

examined.Different brands of tape vary in their ability to resist staining procedures without crinkling or becoming opaque and a‘trial-and-error’ approach is needed to identify suitable products from amongst those available locally. Scotch tape (3M, St. Paul, MN, USA) and Sellotape   Diamond Clear (Henkel Ltd., Hemel Hempsted, UK) often work well.

实用性。胶带粘贴是一种快速的、通用的获取角质层细胞及其微生物的方法，在兽医临床中得到了广泛接受。它也已用于许多治疗研究，但这些出版物应该讨论该方法的局限性。可以将胶带粘贴到难以直接滑动的深折或凹陷区域，通常不需要保定动物。粘合特性有助于去除皮肤表面物质，尤其是在少量渗出的红斑病变中。通过对胶带样品中一定数量的显微镜视野中的酵母菌细胞进行计数，检查了皮肤表面已知区域。不同品牌的胶带在不引起皱纹或不透明的情况下抵抗染色过程的能力各不相同，需要反复试验从本地可用的产品中找出合适的。3M透明胶带和钻石级透明胶带通常效果好。

Figure 3. Tape-strip impression from the lip fold of a dog with Malas

sezia pachydermatis dermatitis.

Abundant ovoid to short cylindrical yeast cells with broad-based budding amongst squames; modifified Wright-Giemsa stain (“Diff-Quik”),x50

图 3.厚皮马拉色菌皮炎患犬唇褶上的胶带采样。

皮屑间有大量卵圆形到短圆柱状酵母细胞，在宽基部出芽；改良的瑞氏吉姆染色剂（“ Diff-Quik”），x50。

8.2.2Impression culture

8.2.2压片培养

Culture of impression samples obtained using contact plates,adhesive tape or Denman brushes for quantification of Malassezia spp. in skin was well described in human dermatology long before veterinarians became interested in this area, although contact plates have been used for the study of cutaneous bacteria in dogs.Squares of sterile carpet, popular for skin surface mycological sampling in France,have also been utilised for recovery of Malassezia spp.in dogs andcats.

早在兽医对该领域感兴趣之前，在人皮肤医学上，已经使用接触板，胶带或Denman刷子获得压片样品的培养物，用于量化皮肤上的马拉色菌，但接触板以用于研究犬皮肤上的细菌。正方形无菌地毯，在法国皮肤表面真菌采样中很流行，在犬猫中也用来马拉色菌采样。

Development. The contact plate technique was shown to be a convenient, rapid and inexpensive method for the quantitative culture of M.pachydermatis from canine skin.Comparable yeast counts were obtained in four serial applications of contact plates containing modified Dixon’s agar to lesional canine skin.When incubated at 32°C, M.pachydermatis colony counts on contact plates after three and seven days of incubation were identical on both modified  Dixon’s agar and Sabouraud’s dextrose agar (SDA), whereas day seven counts on both media were higher than day three counts when incubated at 26°C.

发展。接触板技术被证明是一种简便、快速、经济的从犬皮肤上定量培养厚皮马拉色菌的方法。用改良的Dixon琼脂的接触板，对犬皮肤病变进行四次接触，获得了可比较的酵母菌计数。在32°C下培养后，改良后的Dixon琼脂和SDA琼脂这两种培养基的第七天计数都高于26°C下培养的第三天计数。

Utility for dogs. The contact plate technique has been used to demonstrate significantly elevated groin populations of M.pachydermatis in atopic dogs.In healthy dogs, increased frequencies of isolation and population sizes of M.pachydermatis were found in the lip and interdigital skin when compared with truncal sites.Malassezia pachydermatis populations varied between anatomical sites in healthy basset hounds, with higher populations on the pinnae when compared with the perineal area. Contact plate counts of M.pachydermatis were reduced by both oral ketoconazole and oral terbinafine in healthy basset hounds,by climbazole shampoo in research beagle dogs, and by chlorhexidine and climbazole wipes in a pilot study of five shar pei dogs.

犬中实用性。接触板技术已被用来证明在特应性皮炎患犬中腹股沟的厚皮马拉色菌菌群明显升高。在健康犬中，唇部和指间皮肤中的厚皮马拉色菌的发生频率和种群大小与躯干部位相比有所增加。健康巴吉度猎犬口服酮康唑和口服特比萘芬，研究的比格犬使用氯咪巴唑香波，以及对五只沙皮犬进行用氯己定和氯咪巴唑湿巾试验研究，接触板厚皮马拉色菌数都下降。

Utility for cats. Contact plate counts from the trunk were significantly higher in healthy Devon rex cats when compared to healthy domestic short-haired cats and Cornish rex cats; seborrhoeic Devon rex and sphynx cats had high yeast counts that exceeded those of the other groups. Contact plates were also used in two studies that assessed the beneficial therapeutic effects of oral itraconazole in cats with Malassezia dermatitis.

猫中实用性。与健康的家养短毛猫和康沃尔卷毛猫相比，健康的德文卷毛猫的躯干接触板数量明显更高；脂溢性皮炎的德文卷毛猫和斯芬克斯猫的酵母菌计数高于其他组。接触板还用于两项研究，评估了口服伊曲康唑治疗马拉色菌性皮炎患猫有效。

8.2.3Dispersal methods–detergent (cup) scrub

8.2.3分散法-洗涤剂（杯）擦洗

Development. A detergent scrub technique using wash fluid comprising 0.075 M phosphate-buffered saline, pH7.9 and 0.1 percent Triton X-100, first reported for studies of the human cutaneous bacterial flora,and subsequently in studies of cattle and dogs,is considered the‘gold standard’method for quantitative culture.The method was also used to quantify Malassezia organisms on human skin.In a series of development studies for quantifying M.pachydermatis on canine skin,counts of the yeast in suspension of pure cultures and in skin washings declined in contact with Triton X-100 and other detergents. Vortexing or manual or mechanical shaking of the samples yielded comparable counts. The technique was judged suitable for the quantitation of M.pachydermatis on canine skin provided samples were processed without delay and preferably within one hour of collection.

发展。一种使用含有0.075M磷酸盐缓冲液、PH7.9和0.1%Triton X-100的洗涤剂擦洗技术，用于人皮肤细菌菌群的研究，随后在牛和犬的研究中被认为是定量培养的金标准方法。该方法还用于定量人皮肤上的马拉色菌。在定量研究犬皮肤上的厚皮马拉色菌的一系列开发研究中，纯培养物悬浮液和皮肤中的酵母菌数量在接触Triton X-100 和其他清洗剂后下降。样品的涡流或手动或机械摇动产生了可比的计数。如果样本及时处理，最好在采集后一小时内处理，该技术被认为适合定量犬皮肤上的厚皮马拉色菌。

Utility. Detergent scrub sampling supported a genetic predisposition to Malassezia overgrowth in basset hounds by demonstrating high populations (compared to healthy mixed-breed dogs) in the axillae of healthy hounds and even higher numbers in the axillae of seborrhoeic basset hounds.Detergent scrub counts were also used to evaluate and compare antimicrobial efficacy of shampoos in the treatment of seborrhoeic hounds.

实用性。洗涤剂擦洗采样，与健康的混血犬相比，通过证明健康猎犬腋窝中马拉色菌数较高，脂溢性皮炎巴吉度猎犬腋窝马拉色菌数更高，从而支持了巴吉度猎犬马拉色菌过度增殖的遗传易感性。洗涤剂擦洗计数还用于评估和比较香波在治疗脂溢性皮炎猎犬中的抗菌效果。

8.2.4Dispersal methods–swab wash

8.2.4分散方法-拭子清洗

Development. Microbial populations recovered using conventional swabs can be quantified by dispersing organisms from the swab tip using solutions containing detergents; counts can be achieved by plating serial dilutions onto relevant culture media. This method was used previously in the quantification of staphylococci from porcine,human and canine skin and/or mucosae.Malassezia pachydermatis counts did not vary significantly in each of four swabs applied serially to anus and external ear canal of healthy beagle dogs; dry swabs and moistened swabs had comparable efficiency at recovering the yeast.Yeast counts were comparable when swab tips held in wash fluid were shaken manually or vortexed for 30 s.

发展。可以通过含有清洁剂的溶液分散拭子头中的生物来量化使用常规拭子回收的微生物种群。可以通过将系列稀释液铺在相关培养基上来实现计数。该方法以前曾用于定量猪、人和犬皮肤和/或粘膜的葡萄球菌。健康的比格犬中，连续涂于肛门和外耳的四支拭子中的马拉色菌计数均无显著差异；干棉签和湿棉签在采集酵母菌方面具有相似效率。手动摇动或漩涡洗涤3秒的棉签头，酵母菌计数相当。

Utility. Using the swab wash technique, the anus was the most frequently colonised site in healthy mixed breed and beagle dogs,whereas M. pachydermatis was more frequently isolated in higher populations at nasal and buccal sites in both healthy and seborrhoeic basset hounds.Swab-wash counts from the claw fold of sphynx cats and seborrhoeic Devon rex cats exceeded those of healthy domestic short haired, Cornish rex and Devon rex cats, whereas ear and anus populations were comparable.

实用性。使用拭子清洗技术，肛门是健康混合品种和比格犬中最常见的定植部位，而健康犬和脂溢性皮炎巴吉度猎犬在鼻和颊部位更常分离到厚皮马拉色菌。斯芬克斯猫和脂溢性皮炎德文卷毛猫的爪褶处拭子计数超过了健康的家养短毛猫、康沃尔卷毛猫和德文卷毛猫，但耳部和肛门数量相似。

8.3Comparison of methods

8.3方法比较

A number of investigators have evaluated the relative effectiveness of different sampling methods for Malassezia yeasts, primarily in dogs rather than cats. One early publication reported a subjective preference for direct impression using glass slides over use of cotton swabs, skin scraping and histopathology. Others found that both tape-strips and dry scrapes yielded high yeast counts from lesional skin whereas moistened swabs were much less efficient.Similarly, various authors have reported that swab cytology was inferior to superficial scraping,tape-stripping and conventional culture of swabs.y contrast, Malassezia spp. yeasts and extracellular cocci were recovered in higher numbers from the dorsal claw fold (following skin eversion) by gentle scraping with the sharp point of a tooth pick, when compared with tape-stripping and direct impression using glass slides, likely reflecting enhanced cell exfoliation from the hard cuticle of the claw surface in this specialised site.

许多研究人员评估了不同采样方法对马拉色酵母菌的主要影响，主要是对犬而非猫。早期的一篇出版物报道说，使用玻片直接压片比使用棉签、皮肤刮片和组织病理学更为主观。其他人发现，胶条法和干擦法在病变皮肤上均会产生较高的酵母菌计数，而湿拭子效率低得多。类似地， 各种作者也报道拭子细胞学检查不如皮肤浅刮、胶带黏贴和传统拭子培养。相比之下，马拉色酵母菌与胶带粘贴和玻璃片直接压片相比，通过刮片并用牙签的尖端从背爪褶皱（皮肤外翻后）中，酵母菌和细胞外球菌数量更高。可能反映了在爪表面的硬角质层特殊部位细胞脱落的增强。

In a study of normal canine skin it was reported that the tape culture method was effective at recovering Malassezia but only low numbers of yeast cells were found in samples prepared by direct glass slide impression, skin scraping or cotton swab sampling.Malassezia pachydermatis was more frequently detected in a group of dogs with chronic dermatitis using a tape culture technique when compared with conventional swabbing. Potential growth interference from volatile antimicrobial components of the tape should be evaluated when this cultural method is utilised.

在对正常犬皮肤的研究中，通过胶带培养法可以有效地获得马拉色菌，但通过直接玻片压片，皮肤刮片或棉签采样制备的样品中仅发现少量酵母细胞。马拉色菌与常规拭子采样相比，使用胶带培养技术在一组慢性皮炎犬中更经常检测到这种情况。当使用这种培养方法时，应评估胶带挥发性抗生素成分的潜在的干扰。

Yeast counts on diseased skin (18 sites in six dogs) measured by using detergent scrubs and a contact plate technique were significantly correlated whereas counts obtained by the tape-strip technique from adjacent skin did not correlate with either quantitative cultural method. Varied results were reported in other studies where tape-strip counts and contact plate counts have been compared. Contact plate counts typically yield M.pachydermatis population densities 100-fold lower than those derived by the dispersal detergent scrub technique  and yeast cell densities measured by tape-strip. Thus, the contact plate technique may have low sensitivity for yeast detection when true populations are very low, but may prove more efficient when yeast are more abundant such as in clinical cases or in selected breeds with high populations in healthy skin.

使用洗涤剂擦洗法和接触板技术测得的患病皮肤（六只犬中的十八个部位）上的酵母计数显着相关，而通过胶条技术从相邻皮肤上获得的酵母计数与这两种定量培养方法均无关。 其他研究报告结果，其中比较了胶带计数和接触板计数厚皮马拉色菌密度比用分散洗涤剂擦洗技术和通过胶带法测量的酵母细胞密度低一百倍。因此，当检测的酵母菌非常低时，接触板技术对酵母菌检测的灵敏度可能较低，但当检测酵母菌含量更高时，例如在临床病例中或在健康皮肤中具有较高种群的特定品种中，可能证明更有效。

8.4Culture media

8.4培养基

In quantitative investigations of the skin flora, the culture medium is known to have signifificant effects on the counts of Malassezia yeasts derived from human skin,prompting analogous canine studies.Amongst Malassezia spp.,M.pachydermatis is the least fastidious and normally grows readily on routine media such as SDA,although M.pachydermatis variants with more-exacting lipid requirements have been isolated.

在对皮肤菌群的定量研究中，已知培养基对源自人皮肤的马拉色酵母菌计数有重大影响，类似情况也出现在犬研究中。在马拉色菌中，虽然已分离出脂质需求更精确的厚皮马拉色菌，但厚皮马拉色菌是最不挑剔的，通常很容易在常规培养基如 SDA上生长。

In early qualitative studies, the addition of 1% Tween 80 to a dextrose/ yeast extract agar enhanced the isolation of M.pachydermatis.Tween 80 was not needed for the selective and differential isolation of M.pachydermatis in the presence of peptone,now recognised as the essential lipid source for growth of that species on SDA.Malassezia pachydermatis was isolated from swab wash samples in comparable numbers after three and seven days of incubation at 32°C on each of five media; SDA, SDA plus 1% Tween 80, Ushijima’s medium A, modified Dixon’s agar and Leeming’s medium.After three days of incubation, the colonies were most distinctive on modified Dixon’s agar, forming buff-coloured domed colonies 1–1.3 mm in diameter that were readily distinguished from other cutaneous microbes; on other media the colonies were low convex. Colony counts on contact plates were significantly greater after incubation for seven days on SDA and modified Dixon’s agar than after three days at 26°C, whereas three day and seven day counts were com- parable at 32°C; this poor growth on SDA at 26°C was in accordance with previous and subsequent reports.Counts from swab-wash samples grown on SDA or modified Dixon’s agar for four or seven days were comparable at 32 and 37°C. When compared with aerobic culture, an atmosphere of 5–10% carbon dioxide significantly increased the frequency of isolation and the colony counts on SDA but not modified Dixon’s agar in swab-wash samples incubated at 37°C for seven days.

在早期的定性研究中，向葡萄糖/酵母提取琼脂中添加1％吐温80可以增强厚皮马拉色菌分离。在蛋白胨存在下对厚皮马拉色菌进行选择性分离和菌种鉴定不需要吐温80，目前认为蛋白胨是厚皮马拉色菌在SDA上生长的必要脂质来源。在五种培养基中（SDA、SDA加1％Tween 80、Ushijima培养基 A、改良的迪克森琼脂和李明培养基），32°C培养三到七天后，从棉签洗涤样品中分离出了的厚皮马拉色菌数量相似。培养三天后，菌落在改良的迪克森琼脂上最显著，形成浅黄色、长度为 1-1.3 mm的圆顶状菌落，易与其他皮肤微生物区分开。在其他培养基上菌落呈低凸。在SDA和改良的迪克森琼脂上培养七天后，接触板的菌落计数显着高于26°C 下 3三天后的菌落数，而 32°C 下 三天和七天的菌落数相似。在 26°C 下 SDA 的这种不良生长与先前和随后的报告一致。在 32°C 和 37°C 下，在 SDA 或改良的迪克森琼脂上生长四或七天的拭子样本的计数相似。与有氧培养相比，在 37°C 下培养七天的拭子洗涤样品中，二氧化碳浓度为 5-10％的空气显着增加了分离的频率和 SDA 上的菌落计数，但改良迪克森琼脂中没有显著增加。

One study compared Malassezia counts on SDA and modified Dixon’s agar from swabs and carpet squares at 27 and 37°C.The frequencies of isolation were significantly higher on modified Dixon’s agar at both temperatures after eight days of incubation; higher counts of larger colonies were cultivated on modified Dixon’s agar.

一项研究比较了在 27°C  和 37°C 下从棉签和地毯方块上的SDA 和改良的迪克森琼脂计数的马拉色菌计数，培养八天后，在两种温度下，改良的迪克森琼脂的分离频率均显着更高；在改良的迪克森琼脂上培养了更高数量的较大菌落。

To the best of the authors’ knowledge, there are no reports of comparative studies on quantification of Malassezia spp. from feline skin. Although M.pachydermatis predominates on both healthy cats and cats with dermatitis, lipid-dependent species such as M.sympodialis,M.globosa,M.furfur, M.nana,and M.slooffiae are occasionally encountered. Modified Dixon’s agar has proven suitable for the isolation of these species at 32°C over a seven day incubation period, whereas SDA does not support their growth.

据作者所知，尚无关于猫皮肤的马拉色菌的定量比较研究的报告。但健康猫和皮炎患猫都以厚皮马拉色菌为主，但脂质依赖性菌种，例如合轴马拉色菌、糠秕马拉色菌、娜娜马拉色菌和斯洛菲马拉色菌偶见。事实证明，改良的迪克森琼脂适合在32°C下培养七天的过程中分离这些菌种，而 SDA不支持其生长。

8.5Recommendations for culture

8.5培养建议

Modified Dixon’s agar is the preferred medium for the isolation and quantification for M.pachydermatis from canine skin in view of the relatively rapid growth of distinctive colonies and its potential for supporting the growth of lipid-dependent isolates; incubation should be aerobic at 32–37°C for at least three days. SDA (preferably supplemented with 1% Tween 80) is an alternative for dogs if modified Dixon’s agar is unavailable, although occasional more-lipid dependent isolates will be overlooked with this medium; temperatures below 32°C should be avoided and use of 5-10% carbon dioxide should be considered.

考虑到独特菌落的相对较快生长及其支持脂质依赖性分离株生长的潜力，改良的迪克森琼脂是从犬皮肤中分离和定量厚皮马拉色菌的首选培养基。在 32–37°C 有氧环境下至少培养三天。如果无法获得改良的迪克森琼脂，SDA（最好添加1％吐温80）是替代选择，但这种培养基有时会忽略更多的脂质依赖性分离株。硬应避免温度低于 32°C，并应考虑使用 5-10％的二氧化碳。

In cats, samples should be grown on modified Dixon’s agar at 32–34°C aerobically; cultures should be systematically extended to at least seven days in case of presence of slower growing Malassezia spp. Temperatures in excess of 34°C must be avoided because of the potential to inhibit the growth of thermosensitive species such as M.globosa known to inhabit feline skin.

在猫中， 样品应在 32–34°C 的有氧环境下在改良的迪克森琼脂上生长；如果出现生长较慢的马拉色菌属种，则应系统地将培养至少延长至七天。必须避免超过 34°C 的温度，因为它有可能抑制热敏感菌种，例如已知猫皮肤中的球形马拉色菌。

8.6Defining normal and abnormal populations of Malassezia spp. yeasts in the context of clinical reports

8.6定义马拉色菌酵母菌在临床报告中的正常和异常量

Veterinary clinicians in speciality clinical practice are accustomed to systematically evaluating M.pachydermatis populations in inflammatory skin lesions in the majority of their canine and feline patients, most often with cytology; they will frequently record“elevated populations” or“overgrowth”in their clinical records. When these observations are subject to critical scientific review (such as when case series or reports are submitted to journals for publication) problems arise because reviewers and editors tend to prefer objective confirmation of such claims. This has resulted in definitions of abnormal populations in terms of “more than XX yeast cells in YY oil-immersion (x100) fields”.

兽医临床医生在专科临床实践中习惯于系统地评估大多数犬和猫炎性皮肤病变中的厚皮马拉色菌种群，最常使用细胞学检查。他们经常在临床中记录“数量增多”或“过度增殖”。当对这些观察结果进行严格的科学审查时（例如当病例分析或报告提交给期刊发表时），就会出现问题，因为审阅者和编辑倾向于对这些主观描述进行客观确认。这导致根据“在YY 油镜（x100）下有XX个以上的酵母菌细胞”出现定义偏差。

It is important to appreciate that these definitions are likely arbitrary unless previous studies established the likely values obtained with the identical sampling method in that particular anatomical site in that particular breed of dog or cat. Marked variations in populations between anatomical sites within the same dog are routinely described; for example, contact plates may yield confluent growth from the lip region of healthy dogs whereas counts are routinely much lower in the axilla and groin. Breed variation is extreme in some circumstances, for example in basset hounds and Devon rex and sphynx cats.It is important to note that high populations can be recovered from clinically normal skin; using the‘gold standard’ detergent scrub samples, population sizes in healthy and seborrhoeic basset hounds were shown to overlap.“High”counts need not necessarily be clinically relevant in each case where they are detected.By contrast, “normal”or at least “lower”populations may be sufficient to exacerbate cutaneous inflammation in patients with immediate or delayed hypersensitivity responses.Ultimately, a commensal existence or cutaneous disease caused by M.pachydermatis involves complex interactions between the yeast and its’ mammalian host that are not simply a result of achieving a particular number or density of yeast cells within the stratum corneum.

重点要意识到，除非先前的研究确定了在特定的猫或犬的特定解剖部位使用相同的采样方法获得的可能值，否则这些定义可能是任意的。经常描述同一只犬的解剖部位之间种群的明显变化。例如，从健康犬的嘴唇区域采样的接触板可能会产生融合生长，而通常腋窝和腹股沟的数量要低得多。在某些情况下，例如在巴吉度猎犬和德文卷毛和斯芬克斯猫中，有品种多样化。重要的是要注意，可以从临床上正常的皮肤中获得高数量样本，使用黄金标准洗涤剂擦洗样本，健康犬和脂溢性巴吉度猎犬的菌量显示有重叠。高计数不一定在每种情况下都与临床相关。通过对比，正常或至少较低的种群可能足以加剧具有速发或迟发的超敏反应，使患者产生皮肤炎症。最终，由厚皮马拉色菌引起的共生存在或皮肤疾病涉及酵母菌与其酵母菌之间的复杂相互作用。哺乳动物宿主，这不仅仅是在角质层内达到特定数量或密度的酵母细胞的结果。

8.7Conclusions: recommendations for skin sampling in dogs and cats

8.7结论：建议对犬猫进行皮肤采样

Routine cytological sampling of skin sites in the veterinary clinic is best achieved by light microscopical examination (50x or 100x oil objectives) of tape-strips or dry scrapes stained with modified Wright Giemsa stain (“Diff-Quik”or generic equivalents). Cytology using swabs is normally best restricted to use in the ear canal. Factors such as important variations in anatomical site, breed, sampling method and host immune status commonly thwart thinterpret population (“XX yeasts in YY fields”); trial therapy is routinely required to establish this (Sections 8.5 and 14.1–3). A single report indicates that the sharp end of a tooth pick is the preferred method for sampling the dorsal claw fold in dogs.

在兽医诊所对皮肤部位进行常规的细胞学取样，最好是通过对胶带或刮片进行光学显微镜检查（50倍或100倍油镜），然后用瑞氏吉姆染色剂（Diff-Quik）染色。用于耳道时，通常最好使用拭子进行细胞学检查。诸如解剖部位、品种、采样方法和宿主免疫状态的重要变化等因素，通常会阻碍解释观察到的种群的临床意义（“ YY 视野中的 XX 个酵母菌”）；为确定这一点，通常需要进行经验性治疗（8.5节和14.1-3节）。一份报告表明，最好使用刀片尖端来给犬背部和爪部皱褶处取样。

Routine cultures provide primarily qualitative data on presence/absence of yeast, although “heavy” growth on primary isolation plates likely indicates a high population.Contact plates allow convenient, rapid and inexpensive quantitative culturing of M.pachydermatis from canine and feline skin; they are suitable for both diagnostic and research purposes. Optimally sized plates for cats and dogs (typically 18–27 mm diameter depending on site sampled) containing the preferred medium (modified Dixon’s agar) are not available commercially but are readily custommade in mycology laboratories that are accustomed to preparing their own media.

常规培养主要提供有关酵母菌存在/不存在的定性数据，但在初级分离板上的大量生长可能表明存在量高。接触板允许从犬和猫的皮肤中方便、快速和经济地定量厚皮马拉色菌。它们适合于诊断和研究目的。包含首选培养基（改良的迪克森琼脂）的最佳尺寸的猫和犬的平板（通常为 18-27 mm 直径，取决于所采样的位置）在市场上无法买到，但易于在真菌学实验室中定制。

Detergent scrub sampling is the‘gold standard’method for quantitative culture, although it is more suited to a research rather than diagnostic environment because it is suitable for only relatively flat skin on cooperative patients and rapid sample processing is required. This is the optimal technique for mycological assessment in therapeutic product development.

洗涤剂擦洗采样是定量培养的“黄金标准”方法，但它更适合用于研究而非诊断环境，因为它仅适合患者的相对平坦的皮肤，并且需要迅速处理样品。这是治疗产品开发中进行真菌学评估的最佳技术。

9. Diagnostic methods: molecular and mass spectrometry identification of yeasts in culture and skin

9.诊断方法：培养物及皮肤中对酵母菌分子和质量的鉴定

The application of molecular biological techniques has underpinned the radical transformation of the taxonomy of the genus Malassezia over the last 30 years with an expansion from two to 18 recognised  species  (Section 2).This section briefly reviews some of the more commonly reported molecular and mass spectrometry techniques that may be encountered in the veterinary literature on Malassezia species.

近 30 年来，分子生物学技术的应用为马拉色菌属分类学的根本转变提供了支持，从两种扩展到十八种。本节简要回顾了一些更常见的有关马拉色菌种的报道，兽医文献中可能遇到的分子和质谱技术。

9.1Electrophoretic karyotyping

9.1电泳核型

Electrophoretic karyotyping involves the separation of intact chromosomal DNA according to its size on an agarose gel, generating specific banding patterns dependent upon the number and size of chromosomes. This technique differentiates between Malassezia species, but not between genotypes within each species.The ready availability of sequence-based methods has largely superceded this method in yeast identification.

电泳核型分析涉及在琼脂糖凝胶上，根据其大小分离完整的染色体 DNA，根据染色体的数量和大小产生特定的条带模式。该技术可区分马拉色氏菌属的种，而不是每个物种内的基因型。基于序列方的现成可用性，已在酵母鉴定中取代了其他方法。

9.2Ribosomal RNA gene sequencing

9.2核糖体 RNA 基因测序

Ribosomal RNA gene sequences have proven attractive targets for molecular fungal taxonomy on account of the presence of multiple copies in the genome and their tendency to have combinations of highly-conserved sequences that may be recognised by panfungal PCR primers, separated by highly varied species-specific or genotype-specific domains. Amongst these, the D1/D2 domain of the 26S rRNA and the internal transcribed spacer (ITS) regions have been frequently used in the identification and phylogenetic studies of Malassezia species (Figures S1 and S2).Partial sequencing of the 26S rRNA of one hundred M.pachydermatis isolates identified seven sequence types, named sequevars (Ia-Ig); some of these variants seem to be host-specific for dog, ferrets and rhinoceros.

核糖体 RNA 基因序列已被证明是分子真菌分类学的有吸引力的靶标，原因是基因组中存在多个拷贝，并且它们倾向于具有高度保守的序列组合，这些序列可能被泛真菌 PCR 引物识别，并被高度不同的物种所分隔特定或基因型特定区域。其中，26S rRNA 的 D1 / D2 结构域和内部转录间隔区（ITS）区域经常被用于鉴定和鉴定马拉色菌种。 局部测序一百的26S rRNA 厚皮马拉色菌鉴定出七种序列类型，称为后继体 （Ia-Ig）；其中一些变体似乎对犬，雪貂和犀牛具有宿主特异性。

Marked sequence diversity of the intergenic spacer (IGS)(Figure S1 and S2) of M. pachydermatis isolated from dogs and cats enabled identification of three major groups (1, 2, 3) with 10 subtypes (1A, 1B, 1C, 1D, 2A, 2B, 3A, 3B, 3C and 3D).In Japan,Korea and Taiwan,isolates of  M.pachydermatis of subtype 3D were obtained more frequently from cAD skin lesions than from healthy canine skin. Moreover,subtype-3D strains isolated from atopic dermatitis skin produced higher amounts of phospholipase A2 than did strains of other genotypes,supporting the hypothesis that the pathogenicity of this yeast is associated with the production of high levels of phospholipase A2.By contrast, lack of sequence diversity in the IGS region of M.nana isolates,and in the ß-tubulin gene and microsatellite profilesindicate that a particular M.nana genotype predominates in cats.

从犬猫分离出的厚皮马拉色菌基因间隔区（IGS）的标记序列多样性使得能够鉴定具有十个亚型 （1A，1B，1C，1D， 2A，2B，3A，3B，3C 和 3D）。在日本、韩国和台湾，从 cAD 皮肤病变中获得的3D亚型厚皮马拉色菌比从健康犬皮肤中获得的频率更高。从特应性皮炎的皮肤分离出的3D亚型菌株比其他基因型菌株产生的磷脂酶 A2 含量更高，支持了这种酵母菌的致病性与高水平磷脂酶 A2 产生有关的假设。相反的是，缺乏娜娜马拉色菌分离株，β-微管蛋白基因和微卫星图谱的IGS区域中的序列多样性分析表明，特定的娜娜马拉色菌基因型在猫中占主导地位。

9.3Chitin synthase 2 and ß-tubulin genes

9.3几丁质合酶2和ß-微管蛋白基因

Amplification of the chitin synthase 2 (CHS2) gene in seven Malassezia species yielded an ~620 bp fragment with 95% sequence homology between the species, although phylogenetic analyses indicated that each species was genetically distinct.Limited genetic variability in seen in the CHS2 gene amongst M.pachydermatis isolates from dogs in previous studies,although wider variation was observed in another study.Partial sequencing of the ß-tubulin gene in Malassezia provides a further opportunity for species and genotype differentiation.

在七个马拉色菌属物种中扩增几丁质合酶2（CHS2）基因产生了一个约620 bp的片段，该物种之间具有 95％的序列同源性，但系统分析表明每个物种在遗传上都是不同的。在以前的研究中，从犬中分离到的厚皮马拉色菌分离株 CHS2 基因中可见的有限遗传变异，但在另一项研究中变化很大。马拉色菌ß-微管蛋白基因的部分测序，给物种和基因型分化提供了进一步的机会。

9.4Multilocus PCR approaches

9.4多基因PCR

Genotypic approaches involving analyses of sequences obtained from multiple loci on the Malassezia genome, such as combined analyses of D1/D2, ITS, CHS2 and ß-tubulin sequences, has proven to be a powerful tool in the epidemiological assessment of the intraspecific variations and adaptation of these particular genotypes to specific hosts.

基因型方法涉及从马拉色菌属基因组上多个基因获得的序列分析，例如 D1/ D2，ITS，CHS2 和 ß-微管蛋白序列的组合分析，已被证明是流行病学评估种内变异和适应性的有力工具这些特定基因型中的特定方法。

9.5Random amplification of polymorphic DNA

9.5随机扩增多形态DNA

As the name suggests, random amplification of polymorphic DNA (RAPD) is a PCR-based method where short primers bind to random sites on the yeast genome to produce fragments that create specific banding patterns on electrophoresis. Whilst reproducibility is sometimes challenging, it is regarded as a useful epidemiological tool amongst Malassezia species. One study reported that RAPD analysis of M.pachydermatis indicated the existence of four different genotypes (genotype 1 to 4), with the genotype-1 strains producing the highest amounts of biofilm and phospholipase activity.Moreover, the study also reported that the inflammatory response induced by M.pachydermatis strains in immortalized human keratinocytes (HaCat cells) was significantly higher in cells co-cultured with strain of genotypes 1 or 2.Conversely, cells infected with strains of genotype 3 or 4 did not significantly induce expression of TLR-2 and cytokines; overall these data further suggest a possible association between genotype and virulence factors expressed by M.pachydermatis strains.

顾名思义，多态 DNA 的随机扩增（RAPD）是一种基于PCR 的方法，其中短引物与酵母基因组上的随机位点结合，以产生在电泳中产生特定条带模式的片段。虽然重现性有时极具挑战性，但它被视为马拉色菌属中的一种流行病学工具。一项研究报告说，对厚皮马拉色菌的 RAPD 分析 表明存在四种不同的基因型（基因型 1-4）。此外，该研究还报告说，在永生化的人角质形成细胞（HaCat 细胞）中，厚皮马拉色菌菌株诱导的炎症反应在共培养的细胞中明显更高。基因型为一或二.相反，细胞感染了基因型三或四个没有明显诱导 TLR-2 和细胞因子的表达；总体而言，这些数据进一步表明，由厚皮马拉色菌菌株表达的基因型和毒力因子之间可能存在关联。

9.6 Next generation sequencing and the microbiome

9.6 下一代基因的测序和微生物组

Methods based on next generation sequencing (NGS) have allowed a better characterization of the complex microbial communities occurring on the skin and made it possible to detect Malassezia species that would other- wise be missed using culture based methods (Section 4.4).

基于下一代测序（NGS）的方法可以更好地表征皮肤上发生的复杂微生物群落，并有可能检测使用基于培养的方法会遗漏的马拉色菌种（4.4节）。

9.7 Multiplex PCR

9.7 多重 PCR

A multiplex PCR method that utilized multiple hybridization probes for six Malassezia species, including M.pachydermatis, was reported to provide sensitive and rapid identification of Malassezia DNA in skin swab samples from both animals and humans.

据报道，利用多重杂交探针对包括厚皮马拉色菌在内的六种马拉色菌的杂交探针的方法很灵敏，快速地鉴定动物和人皮肤拭子样品中的马拉色菌DNA。

9.8 MALDI-TOF mass spectrometry

9.8 MALDI-TOF 质谱

Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a tool for identification of microbial cells or cell extracts.The method is rapid and sensitive, although identification of isolates is possible only if the spectral database contains fingerprints of the relevant species and strains. Mass spectrometry has also been utilised for the identification of Malassezia yeasts isolated from cases in three French university hospitals. A MALDI-TOF database of main mass spectra has been developed to allow the rapid identification of 14 Malassezia species (Section 2).

基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）已成为鉴定微生物细胞或细胞提取物的工具。该方法快速且灵敏，但仅当光谱数据库包含相关物种和菌株的指纹时，才可能分离鉴定。质谱法也已用于鉴定从三所法国大学医院的病例中分离出的马拉色酵母菌。已建立主要质谱图的 MALDI-TOF 数据库，可快速识别十四种马拉色菌。

9.9 Conclusions

9.9 结论

Molecular techniques are pivotal in the accurate identification of many of the currently recognised Malassezia species, with the usual exception of M. pachydermatis (which is readily distinguished from the other species by growth on Sabouraud’s dextrose agar).In particular, sequencing of D1/D2 domain of the large subunit of the rRNA gene, ITS, IGS, CHS2 and ß-tubulin genes or regions allows for accurate identification of species and recognition of genotypes that may have relevance for host-adaptation and virulence. Microbiome studies utilising next-generation sequencing have the potential to redefine the microbial ecology of mammalian skin. Multiplex PCR and MALDI-TOF MS hold promise for rapid and specific identification of Malassezia from skin and culture specimens, respectively.

分子技术对于准确鉴定许多目前公认的马拉色菌属至关重要，而常见的厚皮马拉色菌除外（通过在 Sabouraud 的葡萄糖琼脂上生长，很容易与其他物种区分开）。特别是，对rRNA 基因大亚基的 D1 / D2 结构域，ITS，IGS，CHS2 和ß-微管蛋白基因或区域进行测序，可以准确鉴定物种并识别可能与宿主适应性和毒力有关的基因型. .利用下一代测序进行的微生物组研究有可能重新定义哺乳动物皮肤的微生物生态。多重PCR和 MALDI-TOF MS 有望分别从皮肤和培养标本中快速，特异性地鉴定马拉色菌。

10.Histopathological features of canine and feline Malassezia dermatitis

10.犬组织学病理特征和猫的马拉色菌皮炎

10.1Background

10.1背景

The histopathological features in skin biopsy specimens from lesions of Malassezia infection in dogs and cats commonly represent an amalgamation of features reflecting both the host response to the yeast as well as the concurrent, underlying disease. Preparation of the skin prior to biopsy and/or the ready loss of surface scale and yeasts during histological processing results in the number of yeasts observed by microscopy to be of questionable interpretative pathological significance. The issue of yeast number is compounded by the potential for hypersensitivity responses to Malassezia allergens to exaggerate the immunological reaction out of proportion to the population density of the yeast (see Section 6). Taken together, a diagnosis of Malassezia dermatitis cannot be made by histopathology alone; it is more realistic to limit

expectations of histopathology to providing evidence of a consistent inflammatory process and/or indication of the nature of an underlying predisposing disorder. In routine cases, skin populations of the yeast are best assessed by cytology or quantitative culture (Section 8). In some cases, histopathological approaches might be best employed after the yeast has been removed by appropriate therapy to enhance the characterisation of underlying disease.

来自犬猫的马拉色菌感染病变的皮肤活检标本的组织病理学特征通常代表特征的混合，既反映了宿主对酵母菌的反应，也反映了并发的潜在疾病。活检之前皮肤的准备和/或组织学处理过程中表面皮屑和酵母菌的容易损失导致通过显微镜观察到的酵母菌数量具有值得怀疑的病理学意义。酵母数量问题与对马拉色菌过敏原的超敏反应可能加剧了免疫反应，与酵母的种群密度成比例，加剧了这种反应。一个不能仅通过组织病理学诊断马拉色菌性皮炎；将组织病理学的期望限制为提供一致的炎症过程和/或潜在易患疾病的性质的指示是更现实的。在常规情况下，最好通过细胞学或定量培养评估皮肤酵母菌数量。在某些情况下，在通过适当的疗法去除酵母以增强潜在疾病的特征后，最好采用组织病理学方法。

10.2Experimental studies

10.2 试验研究

Cutaneous responses to the application of viable and killed Malassezia have been assessed both clinically and histologically in laboratory animals (guinea pigs, mice, rabbits) and humans, primarily with “lipid-dependent” Malassezia species isolated from humans (whose nomenclature pre-dates current revised taxonomy) applied under occlusion.Lesions generally comprised focal areas of scaling that most often resolved without treatment upon discontinuation of inoculation  and were characterised histologically by yeast cells or hyphae in the stratum corneum, variable epidermal hyperplasia and superficial dermal inflammation with granulocytes and lymphocytes. Interestingly, Swiss white mice developed skin lesions whereas the nude (nu/nu) mouse, the hairless mice and nude rats did not.Inoculation of suspensions of M.pachydermatis into the middle ear and dermis of immunosuppressed mice led to transient infection that resolved within 21 days.

已经在实验动物（豚鼠、小鼠、兔子）和人类中，主要是从人中分离出了“脂质依赖性”马拉色菌种（其命名法为最新的），已在临床和组织学上评估了对存活和杀死的马拉色菌应用的皮肤反应。病变通常包括皮屑病变区域，多数情况下在中断接种后无需治疗即可恢复，并在组织学上以酵母菌细胞或角质层中的菌丝，不同程度表皮增生和浅表真皮炎症伴有粒细胞和淋巴细胞。有趣的是，瑞士白色小鼠出现了皮肤病变，而裸小鼠（nu / nu）、无毛小鼠和裸大鼠则没有。免疫抑制小鼠的中耳和真皮中接种厚皮马拉色菌皮肤病的悬浮液，会导致短暂感染在二十一天内恢复。

In laboratory beagle dogs application of M.pachydermatis was associated with epidermal hyperplasia, occasionally with parakeratosis, superficial perivascular dermal inflammation with primarily neutrophils and  lymphocytes, and sometimes mast cells (but not eosinophils); features were more severe at sites that were occluded.Histological changes markedly reduced within seven days of withdrawal of yeast challenge.

在用与研究厚皮马拉色菌与表皮增生相关性的实验室比格犬中，偶见角化不全，浅表血管周真皮层炎症反应，主要是中性粒细胞和淋巴细胞，有时还有肥大细胞（但不是嗜酸性粒细胞），封闭的部位特征更明显。酵母菌激发停止后七天内组织学变化明显减少。

10.3Canine Malassezia dermatitis

10.3犬马拉色菌皮炎

Histopathologically, canine Malassezia dermatitis presents with variable but often marked hyperkeratosis and multifocal parakeratotic hyperkeratosis with the characteristic budding, bottle-shaped yeasts and often cocci (if not lost during processing) over an irregular spongiotic epidermal hyperplasia that extends to the follicular infundibula, see reviews.Exocytosis of lymphocytes is a regular feature and neutrophilic or eosinophilic exocytosis may create epidermal microabscesses. Mast cell exocytosis is rare but a linear array of mast cells at the dermoepidermal junction is not infrequent. Pigmentary incontinence is occasionally observed. The follicular ostia and infundibulae may be dilated with hyperkeratosis, occasional focal parakeratosis and yeast cells, with variable cocci bacteria. The superficial dermis has variable oedema with interstitial and perivascular inflammation of lymphocytes (usually the dominant cell type), plasma cells, histiocytes, neutrophils and eosinophils.

从组织病理学上看，犬马拉色菌性皮炎表现为多变，通常明显的角化过度和多灶性角化不全性角化过度，伴有出芽、瓶状酵母菌，和常见球菌特征（如果处理过程中未丢失），并且在不规则的海绵状表皮增生，延伸到毛囊漏斗部。淋巴细胞的外泌是常规特征，嗜中性或嗜酸性细胞外泌可能会产生表皮微脓肿。肥大细胞外泌很少，但在表皮真皮交界处线性排列的肥大细胞并不少见。偶见色素失禁。毛囊口和漏斗部可能会因角化过度扩张，偶见局灶性角化不全和酵母菌细胞以及数量不等球菌。浅表真皮层具有不同程度水肿，伴有淋巴细胞（通常为主要细胞类型）、浆细胞、组织细胞、嗜中性粒细胞和嗜酸性粒细胞的间质和血管周炎症反应。

As mentioned above, it is important to recognise that yeast cells are not always observed; Malassezia cells  were absent in 23 out of 86 cases in one study.The follicular ostia/ infundibulae may be the best place to find yeast cells when numbers are low. Whilst visible in haematoxylin and eosin stained slides, periodic acid-Schiff or Grocott’s-Gomori methenamine silver stains accentuate the presence of the yeast.

如上所述，重点是并非总是观察到酵母菌细胞。在一项研究中，在八十六例病例中，有二十三例中没有马拉色菌细胞。毛囊口/漏斗部可能是当酵母菌细胞数量少时寻找的最佳地点。在苏木精和伊红染色的玻片上可见，但高碘酸希夫或格罗科特的高莫丁胺银染可以使酵母菌变明显。

Authors in one study proposed standardized histological criteria for the ‘presumptive’or final histopathological diagnosis of‘Malassezia dermatitis’ comprising:

一项研究中，作者提出了标准化的组织学方法，马拉色菌性皮炎的推测性或最终组织病理学诊断的逻辑标准包括：

1.pronounced irregular epidermal and infundibular hyperplasia;

2.prominent epidermal and infundibular parakeratotic hyperkeratosis;

3.diffuse epidermal and infundibular intercellular oedema (spongiosis);

4.diffuse epidermal and infundibular lymphocytic exocytosis;

5.superficial perivascular to interstitial dermatitis wherein lymphocytes are a prominent inflammatory cell type.

6.presence of unipolar budding yeast in the surface and/or infundibular keratin.

1.明显的不规则表皮层和毛囊漏斗部增生；

2.明显的表皮层和毛囊漏斗部角化不全性角化过度;

3.弥漫性表皮层和毛囊漏斗部细胞间水肿（海绵样水肿）；

4.弥漫性表皮层和毛囊漏斗部淋巴细胞外泌；

5.浅表血管周到间质性皮炎，其中淋巴细胞是主要的炎症细胞类型。

6.表面和/或毛囊漏斗部角质中存在单极发芽酵母菌。

In the case of absence of budding yeasts, those authors regarded the histopathological diagnosis as “presumptive”provided the first five criteria were fulfilled. Unfortunately, similar features may be observed in skin biopsies from lesional skin in dogs with cAD,a disease whose signs may mimic or complicate those of canine Malassezia dermatitis. Features more supportive of Malassezia dermatitis include scalloping of epidermal and follicular margins, eosinophilic pustules and diffuse lymphocytic exocytosis, but none of these are pathognomonic.

在没有发现发芽酵母菌的情况下，以上这些特征作者将组织病理学诊断为疑似，前提是满足前五个标准。不幸的是，在患有 cAD 的犬的病变皮肤的皮肤活检中可能观察到类似的特征这种疾病的症状可能模仿或复杂了犬马拉色菌性皮炎的症状。更支持马拉色菌性皮炎的特征包括表皮层和毛囊边缘的扇形，嗜酸性脓疱和弥漫性淋巴细胞外泌，但这些都不是特异性诊断。

10.4Feline Malassezia dermatitis

10.4 猫马拉色菌皮炎

The histopathogical features associated with Malassezia dermatitis in cats are poorly defined although hyperkeratosis and irregular epidermal hyperplasia have been observed in association with abundant yeasts in the stratum corneum, analogous to canine features.The lack of description of the histopathology associated with these yeast in allergic and seborrheic cats that are otherwise healthy reflects infrequent biopsy sampling in cases more readily assessed by cytological techniques or culture.By contrast, more reports are available in cats with generalised infections that are often associated with visceral neoplasia or other serious internal diseases (Table S3).

虽然猫马拉色菌皮炎相关的组织病理学特征定义尚不清楚，但已发现角化过度和不规则的表皮增生，伴有角质层中大量酵母菌，与犬特征相似。在过敏症和脂溢性疾病患猫中缺乏与酵母菌相关的组织病理学描述，这表明通过细胞学技术或培养更容易评估的情况下很少进行活检采样。相比之下，更多的报道涉是关于全性身感染疾病患猫，这通常与内脏肿瘤或其他严重的内科疾病相关。

10.5Other conditions where Malassezia yeasts may be observed histologically

10.5 可以通过组织学观察到马拉色酵母菌的其他疾病

Malassezia yeasts may proliferate opportunistically in response to skin disease and it is therefore not surprising that they may be observed in the stratum corneum in a variety of conditions. According to a standard textbook, Malassezia may feature in cases of bacterial intertrigo, feline hypersensitivity reactions, acrodermatitis of bull terrier dogs, atopic dermatitis, congenital follicular parakeratosis, facial dermatitis of Persian and Himalayan cats, familial paw pad hyperkeratosis, feline acne, feline paraneoplastic alopecia, feline thymoma-associated exfoliative dermatitis, female hyperoestrogenism, food allergy, hypothyroidism, ichthyosis, interdigital furunculosis, mucinosis, nasodigital hyperkeratosis, sarcoptic mange,seborrhoeic dermatitis, Sertoli cell tumour-associated skin disease, superficial necrolytic dermatitis and zinc-responsive dermatosis (Table S4).

马拉色酵母菌可以因皮肤病而条件性增殖，因此在各种疾病中都可以在角质层中观察到。根据标准教科书，马拉色菌病可能是细菌性间擦疹、猫过敏性疾病、斗牛犬肢端皮炎、特应性皮炎、先天性毛囊角化不全病、波斯猫和喜马拉雅猫面部皮炎、家族性爪垫角化过度、猫痤疮、猫副肿瘤性脱毛、猫胸腺瘤相关的表皮剥脱性皮炎、雌激素过多症、食物过敏、甲减、鱼鳞病、趾间疖病、黏蛋白增多症、鼻部趾部角化过度、犬疥螨病、脂溢性皮炎、与支持细胞肿瘤相关的皮肤病、浅表性坏死性皮炎和锌反应性皮炎（表S4）。

10.6Conclusions

10.6 结论

A diagnosis of Malassezia dermatitis cannot be made by histopathology alone. Histopathological features in dogs often comprise hyperkeratosis or parakeratosis, irregular epidermal hyperplasia and spongiosis that extends to hair follicle infundibulae, lymphocyte and granulocyte exocytosis, and a mixed, predominately lymphocytic, superficial perivascular or interstitial infiltrate with variable superficial dermal oedema. Yeast cells may or may not be observed in surface or infundibular stratum corneum (cytology or quantitative culture are preferable for assessment of populations). In cats, histopathological features vary markedly according to the nature of the underlying disorder.

不能仅通过组织病理学来诊断马拉色菌性皮炎。犬组织病理学特征通常包括角化过度或角化不全，不规则的表皮增生和海绵水肿，扩展到毛囊漏斗部，淋巴细胞和粒细胞外泌，以及混合的，主要是淋巴细胞的浅表血管周或间质浸润，并伴有不同程度浅表真皮层水肿。酵母菌细胞可能在角质层表面或毛囊漏斗角质层中观察到，也可能观察不动（细胞学或定量培养可用于评估数量）。在猫中，根据潜在疾病的性质，其组织病理学特征明显不同。

11.Antifungal susceptibility testing and resistance

11.抗真菌药敏试验和耐药性

11.1Introduction

11.1 介绍

Antifungal chemotherapy is widely used in the treatment of a range of clinical presentations in both humans and animals that are attributable to cutaneous Malassezia. Previous reports have highlighted varied in vitro drug susceptibility results amongst the different species and within genotypes of selected species.The need for surveillance of antimicrobial resistance (AMR) in general has increased in parallel with the emergence of AMR as a significant threat in both human medicine and animal health. This section reviews literature relevant to Malassezia spp. and identifies areas for future development.

抗真菌治疗在人和动物中广泛用于治疗一系列因皮肤马拉色菌病产生的临床表现。以前的报道强调了不同物种之间以及所选物种的基因型内不同的体外药物敏感性结果。随着抗菌耐药性（AMR）的出现成为对人类医学和动物健康的重大威胁的同时，监测AMR的需求总体上也有所增加。本节回顾与马拉色菌属有关的文献，并确定未来的发展领域。

11.2Methods

11.2 方法

The susceptibility test is critical for the selection of treatment strategy and the control of drug-resistant pathogens and in circumstances where the organism’s susceptibility cannot be reliably predicted by its’ identity. In 2008, the Clinical and Laboratory Standards Institute published the most recent document (CLSI M27-A3) in its’series of reference standards for susceptibility testing of yeasts.However, these focused on invasive human pathogens such as Candida and Cryptococcus spp. but M. pachydermatis does not exhibit sufficient growth in the standard defined (and lipid-free) RPMI 1640 medium to permit use of the reference method for this species. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) analogous reference susceptibility test for pathogenic yeasts and moulds also specified use of an RPMI-1640 based culture medium that is unsuitable for Malassezia spp. Establishment of reference susceptibility tests for M.pachydermatis may ultimately depend on work by organisations such as the International Society for Human and Medical Mycology (ISHAM) working groups or the Veterinary Committee on Antimicrobial Susceptibility Testing (VetCAST), a subcommittee of EUCAST. In the absence of standard methods appropriate for M.pachydermatis the CLSI M27-A2 and A3 proto- cols have been adapted by various modifications of the process (Table S5).Technical specifics were usefully reviewed in detail, including a discussion of the effects of medium and conditions of incubation, lipid supplementation, inoculum preparation, end-point determination and quality control aspects.

药敏试验对于选择治疗策略和控制耐药病原体以及在无法通过其身份可靠地预测生物敏感性的情况下至关重要。2008 年，临床和实验室标准协会在其用于酵母菌敏感性测试的参考标准系列中发布了最新文件。 然而，这些文件的重点是针对侵入性人类病原体，例如念珠菌和隐球菌，但是厚皮马拉色菌在标准定义（且无脂质）的 RPMI1640培养基中，未表现出足够的生长，无法对该种使用参考方法。欧洲委员会对致病性酵母菌和霉菌的药敏试验（EUCAST）类似的参考药敏试验，也规定使用不适合于马拉色菌的基于 RPMI-1640 的培养基。建立厚皮马拉色菌皮炎的药敏测试可能最终取决于诸如国际人类和医学真菌学协会（ISHAM）工作组或 EUCAST 小组委员会兽药敏感性委员会兽医委员会（VetCAST）的工作。在缺乏适用于厚皮马拉色菌的标准方法的情况下，CLSI M27-A2 和 A3 方案已通过对工艺技术细节 进行了有益的详细审查，包括对培养基和培养条件、脂质补充、接种物制备、终点测定和质量控制方面的影响。

Modified Etest protocols and conventional disc diffusion (CLSI M44-A) have also been used to determine the in vitro susceptibility of M.pachydermatis (Tables S5 and S6); tests of this type are more suited to routine diagnostic use than broth dilution methods. The CLSI- recommended medium (Mueller-Hinton agar supplemented with glucose and methylene blue) optimally supports the growth of M.pachydermatis provided the inoculum is incubated with a lipid source (Tween 40 or 80); agar diffusion using clotrimazole and miconazole discs on this agar yielded values that correlated with MIC determined by broth dilution. Etest, a commercially- available manual in vitro diagnostic method that utilizes a plastic strip with a predefined gradient of antimicrobial agent, was used to establish MIC values for ketoconazole and itraconazole that reportedly correlated well with those obtained using a broth dilution method.One limitation is that strips are not available for all antifungal agents used in dogs and cats.

改良的 Etest 方案和常规的椎间盘扩散术（CLSI M44-A）也已用于确定厚皮马拉色菌皮炎的体外药敏性。这种类型的测试比肉汤稀释方法更适合常规诊断。推荐的培养基（补充了葡萄糖和亚甲基蓝的 Mueller-Hinton 琼脂）可以最佳地支持厚皮马拉色菌的生长，前提是接种物需与脂质一起培养来源（吐温40 或 80);使用克霉唑和咪康唑帖片在琼脂上进行琼脂扩散得到的值与通过肉汤稀释确定的 MIC 相关。Etest 是一种可购的手动体外诊断方法，该方法利用带有预定梯度的抗菌剂的塑料进行建立酮康唑和伊曲康唑的 MIC 值据报道与使用肉汤稀释法获得的 MIC 值具有很好的相关性。一个局限性是，并非犬和猫中使用的所有抗真菌剂都具有这种帖片。

11.3Breakpoints: clinical versus epidemiological

11.3 断点：临床与流行病学

A breakpoint is defined by EUCAST as“specific values of parameters such as minimum inhibitory concentrations (MICs) or inhibition zone diameters on the basis of which microbes can be assigned to the clinical categories “susceptible (sensitive)”, “intermediate” and “resistant”.There is scope for confusion around use of the term breakpoint. “Clinical breakpoints”indicate to the clinician the likelihood of therapeutic success when using a particular antimicrobial against an infectious agent. “Epidemiological cut-off values”(ECOFFs) (syn.epidemiological or microbiological breakpoints) represent the highest acceptable MIC value of the wild-type microbial populations with no acquired phenotypically detectable resistance mechanism; it is important to note that this might not be clinically relevant if the MIC is still below that achievable with the drug at any given site of infection. Unfortunately, interpretive breakpoints for antifungal susceptibility testing of Malassezia species are not yet available; thus, any classification of isolates into susceptible, intermediate and resistant categories remains speculative. This may be especially challenging in the context of topical therapy where high local concentrations are often achieved. Review of existing literature on M.pachydermatis susceptibility testing suggest that, in some cases, previous use of the term“resistant”would be best regarded as an ECOFF rather than a clinical breakpoint although further studies are required to confirm this hypothesis.

断点由 EUCAST 定义为“参数的特定值，例如最小抑菌浓度（MIC）或抑菌圈直径，根据这些参数微生物可划分为临床类别易感（敏感），中度和耐药，术语断点的使用存在混淆的范围。临床断点向临床医生指示当使用针对感染剂的特定抗菌剂时治疗成功的可能性。临界值（ECOFFs）（流行病学或微生物学的折点）代表了没有获得表型可检测到的耐药机制的野生型微生物种群的最高可接受 MIC 值 ；重要的是要注意，如果 MIC 仍低于该药物在任何特定感染部位所能达到的 MIC。不幸的是，尚无用于描述马拉色菌属物种的抗真菌药敏试验的解释性断点；因此，任何分类将分离物的阳离子分为易感，中等和耐药类别仍然是推测性的。这在经常达到较高局部浓度的外部治疗的情况下可能尤其具有挑战性。审查关于厚皮马拉色菌药敏试验的现有文献表明，在某些情况下，最好先使用耐药一词尽管需要进一步研究以确认这一假说，但它被认为是ECOFF 而非临床断点。

11.4Antifungal drug susceptibility testing inMalassezia species

11.4 美国在马拉色菌属的抗真菌药敏试验

11.4.1Azole antifungal drugs

11.4.1 唑类抗真菌药

Imidazoles such as clotrimazole, climbazole and miconazole are used topically (most commonly in otic products, creams or shampoos) although ketoconazole and its triazole derivative itraconazole are widely available for oral use in dogs and cats. Posaconazole, a triazole structurally similar to itraconazole, is used orally or intravenously for the treatment of invasive mycoses in immunocompromised humans,and topically in dogs with Malassezia otitis. Clinical efficacy with these products corresponds to multiple, systematic reports of low MICs in vitro with drugs of this class, with the exception of fluconazole (see below).

虽然咪唑类药物（例如克霉唑、氯咪巴唑和咪康唑）能外部使用（最常见于耳药、软膏或香波），但酮康唑及其三唑衍生物伊曲康唑可广泛用于犬和猫的口服使用。泊沙康唑是一种结构相似于伊曲康唑的三唑药物，可口服或静脉注射用于免疫功能低下的人的侵袭性真菌病，以及外部治疗马拉色菌性耳炎患犬。这些产品的临床疗效对应于多种此类药物的体外低 MIC 的系统报告，氟康唑除外（见下文）。

11.4.2Terbinafine

11.4.2 特比萘芬

Terbinafine is a synthetic allylamine derivative that inhibits fungal ergosterol biosynthesis at the point of squalene epoxidation. Terbinafine was first shown to have activity in vitro against Malassezia spp. more than 30 years ago,although in the same study terbinafine activity was 20–800 times greater amongst a range of dermatophytes. Subsequent to conflicting reports of efficacy using topical versus oral dosing in human pityriasis versicolor, in vitro studies showed lower MICs in isolates of M.sympodialis that correlated with efficacy of oral dosing; higher MICs in M.globosa, M.restricta, M.slooffiae and M.furfur sensu stricto were associated with better responses to the higher concentrations achieved by topical therapy. These species-related differential susceptibilities were confirmed in a subsequent study, with M.sympodialis and M.pachydermatis showing greatest susceptibility amongst examples of relative tolerance amongst other Malassezia species.

特比萘芬是一种合成的烯丙胺衍生物，可在角鲨烯环氧化时抑制真菌麦角固醇的生物合成。特比萘芬在30多年前，首先被证明具有体外抗马拉色菌的活性，但在同一研究中，特比萘芬的活性在一系列皮肤癣菌中高出 20-800 倍。关于在人类花斑癣使用外用和口服给药的疗效相互矛盾的报道之后，体外研究显示，分离出合轴马拉色菌株的 MIC 较低与口服给药功效相关；球形马拉色菌、限制马拉色菌、斯洛菲马拉色菌和糠秕马拉色菌中较高的 MIC 与通过外部治疗获得的更高浓度的更好疗效相关。随后的研究证实了这些菌种相关的不同敏感性，在其他马拉色菌属的相对耐受性，合轴马拉色菌和厚皮马拉色菌表现出最大的敏感性。

Further studies in collections of field isolates of M. pachydermatis have routinely indicated susceptibility at concentrations similar to those obtained with azole antifungal drugs.In a study of 24 M.pachydermatis isolates (urea broth dilution), mean MIC values of terbinafine were favourably comparable to those of itraconazole, although ranges or MIC90 values were not reported.Similar activity was found (supplemented RPMI 1640 medium) with a single M.pachydermatis strain. In a study of 62 canine derived M.pachydermatis strains (Christensen’s urea broth, supplemented RPMI 1640), terbinafine was superior (based on MIC90) to fluconazole by six two-fold dilutions but inferior to ketoconazole and itraconazole by three and five dilutions respectively.These values were barely exceeded in a pharmacokinetic study of skin concentrations achieved after oral dosing at 30 mg/kg sid. In another study (Sabouraud’s broth with 1% Tween 80) of 216 colonies of M.pachydermatis obtained from 28 dogs with otitis (n = 25) or dermatitis (n = 3), terbinafine was superior (based on MIC90) to fluconazole by five two-fold dilutions but inferior to ketoconazole and itraconazole by one and four two-fold dilutions respectively.A similar relationship between terbinafine and ketoconazole susceptibility was noted in a study (supplemented Sabouraud’s broth) of 51 M.pachydermatis strains from canine ears.

在对厚皮马拉色菌田间分离株的收集中进行的进一步研究已常规表明，其敏感性与用唑类抗真菌药获得的浓度相似。在一项针对 24 株厚皮马拉色菌（尿素肉汤稀释液）的研究中，特比萘芬的平均 MIC 值与伊曲康唑的平均MIC 值相当，但未报道其范围或 MIC90 值。发现了类似的活性（补充 RPMI 1640 培养基）在对 62 个犬源性厚皮马拉色菌菌株（克里斯滕森的尿素肉汤，补充 RPMI 1640）进行的一项研究中，特比萘芬（以 MIC90 为基础） 六次两倍稀释后优于氟康唑，但效果较差分别以 3 和 5 倍 的稀释度对酮康唑和伊曲康唑进行稀释 。在口服剂量为 30 mg / kg sid 的皮肤浓度的药代动力学研究中，这些值几乎未超过。在另一项研究中（吐温为 1％的吐温肉汤）从 28 只患有耳炎（n = 25）或皮炎（n = 3）的犬中获得的 216 个厚皮马拉色菌菌落中有 80 个，特比萘芬经5个两倍稀释后（以 MIC90 为基准）优于氟康唑，但次于酮康唑 336 一项对来自犬耳的 51 株厚皮马拉色菌菌株的研究 （补充的 Sabouraud 肉汤）中发现特比萘芬和酮康唑的药敏性相似。

11.4.3Nystatin

11.4.3 制霉菌素

The polyene cyclic macrolides, amphotericin B and nystatin, were amongst the earliest broad-spectrum antifungals introduced for clinical use.The potential toxicity of amphotericin B generally limits its use in veterinary medicine to serious progressive or disseminated systemic mycoses, whereas nystatin is active when applied topically.Its mode of action is via altered cell membrane permeability mediated by preferential binding to ergosterol. These drugs were regarded as reference agents in early studies of M.pachydermatis susceptibility to azoles because of well-established activity against the genus,although polyene-resistant strains have been induced in vitro by exposure to mutagen (N-methyl-N’-ni- trosoguanidine) or UV irradiation.

多烯环大环内酯类两性霉素B和制霉菌素是最早引入临床的广谱抗真菌药。两性霉素B的潜在毒性通常将其限制在兽药中用于严重的进行性或弥散性全身性真菌病，而制霉菌素则为其作用方式是通过与麦角固醇优先结合而介导的细胞膜通透性改变。这些药物被认为是由于对真菌的充分确定的活性，因此对厚皮马拉色菌对唑类药物的敏感性的早期研究表明，但通过诱变剂（N- 甲基-N'-亚硝基胍）或紫外线辐射体外诱导了多烯抗性菌株。

Wild type M.pachydermatis strains appear routinely susceptible to nystatin, although higher MICs (MIC90 increased by a single two-fold dilution) have been reported amongst isolates obtained from dogs with skin disease when compared to isolates from healthy dogs (reportedly RPMI1640).In a study of 51 strains from canine ears (supplemented Sabouraud’s broth), nystatin was inferior to ketoconazole and terbinafine by seven and four two-fold dilutions, respectively.

野生型厚皮马拉色菌菌株似乎通常对制霉菌素敏感，但与健康犬的分离株相比，据报道从患有皮肤病的犬中分离得到的 MIC 较高（MIC90 通过单倍两倍稀释增加）（据报道为 RPMI1640）。在对来自犬耳道的 51 株菌株（补充 Sabouraud 肉汤）进行的研究中，制霉菌素的稀释度分别为酮康唑和特比萘芬的七倍和四倍的双重稀释。

11.4.4Chlorhexidine

11.4.4 氯己定

Previous studies of the activity of chlorhexidine against M.pachydermatis have primarily utilized dilutions of commercial products rather than conventional reference methods with the drug in its’pure  form.Although most of these studies have evaluated very small numbers of isolates, a common theme is that M.pachydermatis is typically susceptible to dilutions of chlorhexidine products, in accordance with reports of clinical efficacy.To the best of the authors’ knowledge, reduced susceptibility to chlorhexidine in vitro has not been reported.

以前关于氯己定对厚皮马拉色菌活性研究主要利用商品化稀释液，而不是常规参考方法，以药物的纯净形式使用。虽然这些研究大多数都评估了极少量的分离物，但一个共同的主题是，典型的厚皮马拉色菌是根据临床疗效的报告，易稀释氯己定产品。据作者所知，体外对氯己定的敏感性降低并未报道。

11.4.5Gentamicin and other aminoglycosides

11.4.5 庆大霉素和其他氨基糖苷类

Gentamicin is an aminoglycoside antibiotic with activity against many aerobic Gram-positive and negative bacteria, commonly formulated in polypharmacy otic products for dogs. Whilst gentamicin has been used as an inhibitor of bacterial growth in cultures of M.pachydermatis from ear swabs from laboratory dogs with high bacterial colonisation, a subsequent report indicated that 20 of 20 isolates were susceptible to high concentrations in vitro. Further studies (Sabourauds agar, Christensen’s urea broth with Tween 40 & 80) have shown a wide variation in effect, with MICs’ of gentamicin ranging from 1.0 to >150 µg/mL.Netilmicin, tobramycin and framycetin also have variable but often high activity against M.pachydermatis.These studies indicate that  gentamicin might not be an optimal drug, for suppression of bacterial growth, in culture media intended for the primary isolation of  M.pachydermatis and that further studies are warranted. The extent of any clinical benefit of anti-Malassezia activity from gentamicin in otic formulations used in canine otitis externa requires further assessment.

庆大霉素是一种氨基糖苷类抗生素，对许多好氧革兰氏阳性和阴性细菌具有活性，通常在犬的复方耳药中配制。虽然庆大霉素已被用作细菌繁殖高的实验犬耳拭子中的厚皮马拉色菌培养物中细菌生长的抑制剂，但随后的报告显示 20种分离菌中的20种在体外对高浓度敏感 。进一步的研究（Sabourauds 琼脂，Christensen 的带有吐温40 和 80 的尿素肉汤）显示了很大的差异，庆大霉素的 MIC 从 1.0 到> 150 µg/mL 不等。奈替米星、妥布霉素和曲霉菌素也具有多样化。这些研究表明，庆大霉素可能不是在旨在初步分离出厚皮马拉色菌的培养基中抑制细菌生长的最佳药物，因此有必要进行进一步的研究。庆大霉素对犬外耳炎使用的耳药中抗马拉色菌活性的任何临床益处的程度都需要进一步评估。

11.4.6 Other drugs

11.4.6 其他药物

A small number of publications report activity in vitro of other agents against Malassezia spp. These include plant-derived substances such as Blad-containing oligomer (an antifungal agent approved for agricultural use); beta-thujaplicin,a honey-based gel,kanuka and manuka and other plant-derived essential oils;agents primarily used in the topical treatment of human dandruff or seborrhoeic dermatitis (selenium sulphide, zinc pyrithione, ciclopiroxolamine， rilopirox and povidone–iodine.The calcineurin inhibitors (tacrolimus and pimecrolimus) have activity in vitro against human-associated Malassezia yeasts.The clinical relevance of these observations for canine and feline infections requires further asessment.

少数出版物报道了其他药物对马拉色菌的体外活性。这些包括植物来源的物质，例如含 叶片的低聚物（已批准用于农业的抗真菌剂）、β-侧柏素（一种蜂蜜基凝胶）、月桂状胶木和麦卢卡和其他植物来源的物质精油; 主要用于外部治疗人头皮屑或脂溢性皮炎的试剂（硫化硒、巯氧吡啶锌、环吡氧胺、培洛克斯）和聚维酮碘、钙调神经磷酸酶抑制剂（他克莫司和吡美莫司）在体外具有抗人相关的马拉色酵母菌的作用。这些观察结果对犬和猫感染的临床意义需要进一步评估。

11.4.7Combinations of antifungal drugs

11.4.7 抗真菌药联用

Combining antifungal drugs might potentiate their efficacy, reduce the selection pressure for resistance, reduce side effects if lower doses can be used and achieve clinical efficacy when monotherapy is ineffective.In a study of 30 M.pachydermatis isolates from canine otitis externa, primarily indifferent and often divergent interactions were observed between azoles and nystatin, terbinafine and caspofungin, although 55% (16 of 30) of isolates showed a synergistic interaction between caspofungin and itraconazole or fluconazole.By contrast, amphotericin B antagonised the activity of itraconazole, but not fluconazole or posaconazole, in approximately 55% of 30 otic isolates of M.pachydermatis in a broth micro-dilution checkerboard system.An additive interaction between fluconazole and calcineurin inhibitors against M.globosa and M.restricta is mediated by the immunophilin gene FKBP12.

联用抗真菌药物可能会增强疗效，降低耐药选择压力，如果可以降低剂量则能降低副作用，并且在单一治疗无效时可达到临床疗效。在一项对30株犬外耳炎分离的厚皮马拉色菌的研究中，唑类和制霉菌素、特比萘芬和卡泊芬净之间作用一般，且通常作用冲突，，但有 55％（30 株中的 16 株）分离株显示卡泊芬净与伊曲康唑或氟康唑之间具有协同作用。相反，在大约55%的30株耳部厚皮马拉色菌分离株的肉汤微稀释棋盘系统中，两性霉素B对伊曲康唑活性有拮抗作用，但对氟康唑和泊沙康唑没有拮抗作用。氟康唑与钙调神经磷酸酶抑制剂对球形马拉色菌和限制马拉色菌的抑制有协同增加作用，是由亲免蛋白基因FKBP12介导的。

11.5Reports of reduced susceptibility to antifungal drugs amongst Malassezia yeasts

11.5 马拉色酵母菌中抗真菌药敏感性降低的报道

11.5.1Selecting for resistance in the laboratory

11.5.1 在实验室中选择性耐药

In a study of 10 selected isolatess of M. pachydermatis from canine ears (supplemented Sabouraud’s broth), terbinafine MIC50 increased by three two-fold dilutions when subcultured 30 times in Sabouraud’s broth containing terbinafine at concentrations close to the original MIC. Similarly, nystatin MIC50 increased by a single two-fold dilution when sub-cultured in the same way and ketoconazole MIC50 increased by four two-fold dilutions. From work in Malassezia and other pathogens, reduced susceptibility to azoles may be caused by reduced affinity due to alterations in the drug target enzyme, by increased expression of the target gene (ERG11) encoding this enzyme, or by increased efflux by overexpression of genes encoding membrane transport proteins of the ABC transporter (CDR1/CDR2) or the major facilitator (MDR1) superfamilies.Induction of high fluconazole MICs in 30 strains of M.pachydermatis by prolonged exposure was associated with elevated MICs to other azoles, suggesting that the molecular basis for these effects may in some cases confer cross-resistance to this drug class.Further studies are needed to define the molecular genetics of the reduced susceptibility to azoles, terbinafine and nystatin induced in M.pachydermatis by laboratory exposure at sub-inhibitory drug concentrations and any implications for long-term drug use in clinical practice.

在一项对 10株从犬耳中选择分离的厚皮马拉色菌菌株（补充 Sabouraud 肉汤）的研究中，当在含有特比萘芬的浓度接近原始 MIC 的Sabouraud 肉汤中亚培养30 次后，特比萘芬 MIC50 增加了三倍的双重稀释度。类似地，当以相同的方式进行亚培养时，制霉菌素 MIC50 会增加一个双重稀释度，而酮康唑 MIC50 则会被四倍的双重稀释度增加。从在马拉色菌病和其他病原体中的工作来看，由于药物靶标酶的变化，编码该酶的靶标基因（ERG11）的表达增加或过表达增加而导致的亲和力降低，可能导致对唑类的敏感性降低编码 ABC 转运蛋白（CDR1 / CDR2）或主要促进因子（MDR1）超家族的膜转运蛋白的基因。长期暴露诱导 30 株厚皮马拉色菌的高氟康唑 MIC 与其他唑类的 MIC 升高有关，这表明这些作用的分子基础在某些情况下可能赋予该药物类交叉耐药性。需要进一步的研究来确定分子遗传学，以降低由帕金森病引起的唑类，特比萘芬和制霉菌素的敏感性。亚抑制药物浓度下的实验室暴露以及在临床实践中对长期使用药物的任何影响。

11.5.2Studies of field isolates

11.5.2 野外分离株研究

Malassezia spp. are reportedly not susceptible to echinocandins and griseofulvin.The inherently lower antifungal potency of the triazole, fluconazole, is largely compensated by its excellent pharmacokinetic properties for deep-seated infections of humans.Amongst collections of M.pachydermatis, fluconazole consistently returns significantly higher MICs when compared with other clinically relevant imidazoles and triazoles,leading to questionable predicted clinical utility of this agent in dogs and cats, pending development of clinical breakpoints and further clinical studies.

据报道，马拉色菌属对棘白菌素和灰黄霉素不敏感。三唑氟康唑固有的较低的抗真菌效力在很大程度上由其出色的药代动力学特性所补偿。在厚皮马拉色菌中，氟康唑与其他临床相关的咪唑和三唑类药物相比，氟康唑的 MIC 始终显着提高，导致该药在犬和猫的临床应用前景可疑，尚待确定临床断点和进一步的临床研究。

In 2010 a study reported the first itraconazole and ketoconazole “resistant”isolate of M. pachydermatis, originating from canine seborrhoeic dermatitis.Subsequently, it was reported that Italian clinical isolates of M. pachydermatis from canine skin lesions had low susceptibility to azoles compared with those from normal canine skin.Another study expressed concern about the phenomenon in the worldwide context, given that secondary infections with M.pachydermatis in cAD are frequently treated using azolecontaining shampoos.These authors investigated azole (itraconazole and ketoconazole) susceptibility amongst M.pachydermatis isolates from cAD cases in East Asia over a two year period(2010 and 2011)by Etest using Sabour-aud’s   dextrose agar supplemented  with 0.5% Tween 40.MICs amongst isolates from cAD  isolates were significantly higher than the isolates from healthy dogs, in parallel to studies of isolates from healthy and seborrhoeic dogs from the same region.Treatment failure in a dog with idiopathic  M.pachydermatis dermatitis that had received years of previously successful treatment using a combination of pulse oral itraconazole and topical therapy was associated with elevated MICs to azoles in vitro.Similar observations were made in studies of M.pachydermatis isolates from dogs with chronic otitis externa. One study reported that most M.pachydermatis isolates from canine otitis externa cases were inhibited by clotrimazole, miconazole and thiabendazole, at concentrations of 2- 8 (clotrimazole), 1–4 (miconazole) or 16–32 (thiabendazole) µg/mL.MICs were higher in M.pachydermatis isolates from chronic otitis dogs that had been previously treated with various topical ear products containing miconazole and clotrimazole.Another study examined in vitro antifungal susceptibility of M.pachydermatis isolated from cases of canine otitis clinically nonresponsive to either topical miconazole or clotrimazole and recognized MICs above the limit of detection for miconazole and clotrimazole in some isolates.Another study reported that high MICs of itraconazole and ketoconazole in a strain of M.pachydermatis from canine dermatitis were associated with missense mutations in the ERG11 gene.An additive effect of itraconazole in combination with tacrolimus likely reflected calcineurin-inhibitor binding to the immunophilin FKBP12, as shown previously for additive interactions with fluconazole in M. furfur and M. sympodialis.

2010年的一项研究报道了首次源自犬脂溢性皮炎的厚皮马拉色菌分离株的伊曲康唑和酮康唑耐药分离株。随后，有报道称意大利临床分离自犬皮肤病变的分离株对霉菌的敏感性低。另一项研究对这种现象在世界范围内表示关注，因为在 cAD 中，经常使用含唑类香波治疗继发性厚皮马拉色菌感染。这些作者研究了唑类药物（伊曲康唑和 Etest 使用 Sabours 葡萄糖琼脂补充了 0.5％的吐温40，在两年内（2010 年和 2011 年）从东亚的 cAD 病例中分离出的厚皮马拉色菌对cAD 病例的敏感性.从 cAD 分离物中分离出的MICs 为与来自同一地区的健康和脂溢性犬分离株的研究相平行，研究结果明显高于来自健康犬的分离株。患有特发性厚皮马拉色菌皮炎犬的治疗失败，该犬已经接受了多年的脉冲口服伊曲康唑治疗联合外部治疗治疗成功，这与体外培养的 MICs 升高相关。在对患有慢性外耳炎的犬中的厚皮马拉色菌分离株的研究中也得出了类似的观察结果。一项研究报告说，从犬外耳炎病例分离的大多数厚皮马拉色菌均被克霉唑，咪康唑和噻苯达唑抑制，浓度为 2-8（克霉唑），1-4（咪康唑）或 16-32 （噻苯达唑）µg / mL。慢性耳炎犬的分离株中的 MICs 较高，之前已用含咪康唑和克霉唑的各种外用耳药治疗过。另一项研究检查了从犬耳炎病例中厚皮马拉色菌分离株的体外抗真菌药性。临床上对外用咪康唑或克霉唑无效，且某些分离株中公认的 MIC 超过了咪康唑和克霉唑的检测限。另一项研究报道，犬皮炎的厚皮马拉色菌中伊曲康唑和酮康唑的高 MICs 与 ERG11 基因的错义突变有关。伊曲康唑与他克莫司联用的疗效增加反映钙调神经磷酸酶抑制剂与亲免蛋白 FKBP12 的结合，如先前在糠秕马拉色菌和合轴马拉色菌中与氟康唑的增加作用所示。

It is important to note that products formulated for treatment of canine otitis routinely contain concentrations of the drug that are at least 650 times the MIC of the wild-type target pathogen. Therefore, antifungal concentrations in topical products typically exceed, by several orders of magnitude, the highest MIC values reported to date for azole-resistant strains.

值得注意的是，配制用于治疗犬耳炎的产品通常含有的药物浓度至少是野生型靶病原体 MIC 的 650 倍。因此，外用药中的抗真菌浓度通常比唑类耐药菌株迄今为止报道的最高 MIC 值高出几个数量级。

11.5.3Biofilms

11.5.3 生物膜

Biofilms, wherein groups of adherent microbial cells become embedded in an extracellular polymeric matrix,may protect the microbe from the host immune system and reduce susceptibility to antimicrobial drugs.One study evaluated the in vitro antifungal susceptibility of M.pachydermatis strains, in both their planktonic and sessile (biofilm production) forms to fluconazole, posaconazole, voriconazole, miconazole, ketoconazole, itraconazole and terbinafine; MIC values were increased by 3–6 two fold dilutions in the sessile form. Another study reported an increase in itraconazole and ketoconazole MICs by nine two-fold dilutions (<0.03 to >16 µg/mL) following biofilm formation in M.pachydermatis,either alone or when cocultured with Candida parapsilosis.A further study also reported that effective concentrations (EC50) of ketoconazole and itraconazole MICs increased by 18–169 and 13–124 times respectively against M.pachydermatis in biofilms when compared to planktonic forms.Another study reported reduced susceptibility of M.pachydermatis biofilms to azoles,terbinafine and amphotericin B.The significance of these important observations for therapy in clinical practice remains to be determined.

生物膜，其中粘附的微生物细胞群嵌入细胞外聚合物基质中，一项研究评估了厚皮马拉色菌菌株的体外抗真菌药性，无论是对浮游型还是固着型（生物膜产生），氟康唑、泊沙康唑、伏立康唑、咪康唑、酮康唑、伊曲康唑和特比萘芬都具有抗真菌作用。 固着型的 MIC 值增加 3–6 倍稀释。另一项研究报道，在厚皮马拉色菌中形成生物膜后，伊曲康唑和酮康唑的 MIC 增加了九倍的双重稀释（<0.03 至> 16 µg /mL）。 进一步的研究还报告说，与生物膜相比，酮康唑和伊曲康唑的 MIC 的有效浓度（EC50）相对于生物膜中的厚皮马拉色菌分别增加 18-169 和 13-124 倍。另一项研究报道了厚皮马拉色菌生物膜对唑类、特比萘芬和两性霉素B的敏感性降低。这些重要观察对临床治疗的意义尚待确定。

11.6 Conclusions: clinical context and implications for practice

11.6 结论：临床背景及其对实践的启示

Testing for resistance is hampered by the unsuitability of the current CLSI and EUCAST reference methods, lack of an agreed modified protocol optimized for M. pachyder- matis and absence of clinical breakpoints for either systemic or topical therapies. Despite these critical limitations, current data suggest that the vast majority of field isolates of M.pachydermatis are routinely susceptible to most relevant azoles (miconazole, clotrimazole, itraconazole, posaconazole and ketoconazole). However, the evidence that reduced susceptibility of M.pachydermatis to commonly used antifungal drugs may develop under both field and laboratory conditions highlights the need for surveillance and vigilance for the emergence of clinically-relevant resistance. This is especially important in cases of cAD, seborrhoeic dermatitis and chronic otitis externa where repeated treatments are commonly utilized. Improved and agreed reference methods designed to overcome the specific growth requirements of Malassezia spp. are therefore urgently required.

当前的 CLSI 和 EUCAST 参考方法的不适用，缺乏针对厚皮马拉色菌的一致认可的改进方案，以及缺乏针对全身或外部治疗的临床断点的耐药性测试，阻碍了耐药性的测试。虽然存在这些重要的限制性，但目前的数据表明，绝大多数的厚皮马拉色菌的现场分离株通常对大多数相关的唑类药物敏感（咪康唑、克霉唑、伊曲康唑、泊沙康唑和酮康唑）。然而，在野外和实验室条件下都可能发展出厚皮马拉色菌对常用抗真菌药敏感性降低的证据，这凸显了对临床相关耐药性出现的监测和警惕的必要性。这对于经常使用重复治疗的 cAD，脂溢性皮炎和慢性外耳炎尤为重要。因此迫切需要经过改进和商定的参考方法，旨在克服马拉色菌的特定生长要求。

喜宝宠物医院