孢子丝菌病43例临床分析

目的 探讨孢子丝菌病的发病原因及特点。方法 就1991～2002年间在本院就诊的深部真菌病病例,利用沙氏真菌学检查方法筛查确诊为孢子丝菌病43例并进行临床观察。结果 1991—1993年患者5例（11．63％）,1994～1996年水灾后2年内患者34例（79．07％）,1997～2002年5年内患者4例（9．30％）。有明显外伤史及疑似蚊、蜂叮螫者占总发病人数72．09％。伴高血压者占20．93％。碘化钾治愈率100％。结论 各种原因造成的环境污染或某些自然灾害致使腐生菌大量生长繁殖是引起本病区域性流行的根本原因。外伤及昆虫叮、螫为重要致病条件。碘化钾为首选治疗药物。     

Influence of Th1/Th2 Cytokines and Nitric Oxide in Murine Systemic Infection Induced by Sporothrix schenckii

In an attempt to elucidate the effects of Sporothrix schenckii infection on the immune response, our laboratory has developed a murine model of disseminated sporotrichosis. Helper T cells can be further subdivided into Th1 and Th2 phenotypes. The differentiation of two subsets of T lymphocytes is driven by IL-12 and IL-4 cytokines, respectively. Th1 cells produce IFN-γ that activate macrophages and promote cell-mediated immunity. In addition, we found low levels of iNOS and NO production in the initial (1st and 2nd weeks) and final (9th and 10th weeks) periods of the infection, in contrast with the period of week 4 to 7 of elevated values. The determination of IFN-γ and IL-12 are in agreement with NO/iNOS detection, showing the presence of cellular immune response throughout the infectious process. However, the production of IL-4 shows an increase in levels after the 5th and 6th weeks suggesting a participation of Th2 response in this period as well. Regarding these results, the study demonstrated that in experimental sporotrichosis infection the cellular immune response participated throughout the period analyzed as a nitric oxide dependent mechanism. In contrast, the presence of Th2 response began in the 5th week, suggesting the participation of humoral immune response in advanced stages of sporotrichosis.     

Current Research on the Immune Response to Experimental Sporotrichosis

Sporotrichosis is often manifested as a chronic granulomatous infection and the monocytes/macrophages play a central role in the host defense system. Surface components of Sporothrix schenckii have been characterized and suggestions have been made as to their possible role in pathogenicity. Ergosterol peroxide, cell-wall compounds (alkali-insoluble fraction-F1 and lipid extract-LEY), and exoantigen from the yeast form of the fungus have been characterized as virulence factors, activating both innate, by cytotoxins linked to the activation of reactive oxygen and nitrogen species (H₂O₂ and NO), and adaptive immune response to produce cytokines Th1 and Th2 profile. In this study, preliminary results have demonstrated that, in systemic sporotrichosis, TLR-4 triggers the innate immune response, activating an oxidative burst. These data represent the first report of the participation of TLR-4 in murine sporotrichosis, in the presence of lipids from the cell wall of S. schenckii. These results taken together may open new perspectives of study leading to an antifungal agent that could be used to benefit the entire population.     

Detection of cellular immunity with the soluble antigen of the fungus Sporothrix schenckii in the systemic form of the disease

Sporothrix schenckii is the etiologic agent of sporotrichosis, a mycosis of world-wide distribution more commonly occurring in tropical regions. The immunological mechanisms involved in the prevention and control of sporotrichosis are not fully understood but apparently include both the humoral and cellular responses. In the present investigation, cellular immunity was evaluated by in vivo and in vitro tests in mice infected with yeast-like forms of S. schenckii. The disease developed systemically and cellular immunity was evaluated for a period of 10 weeks. The soluble antigen utilized in the tests was prepared from yeast form of the fungus through the sonication (20 min: 10 sonications at 50 W at 2-min intervals). Delayed hypersensitivity and lymphocyte transformation tests showed that the cellular immune response was depressed between the 4th and 6th week of infection when the animals were challenged with the soluble fungal antigen. This depression frequently indicates worsening of the disease, with greater involvement of the host. This is a promising field of research for a better understanding of the pathogeny of this mycosis.     

Sporotrichosis with formation of sporotrichids

Summary A case of sporotrichosis with sporotrichid is presented. Satisfactory cultures were obtained only after animal inoculation. Cure resulted from use of Organidin, an organic form of iodine, when a severe sensitivity occurred to KI. The fact that sporotrichosis has many clinical forms is emphasized.

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Résumé Un cas de sporotrichose, avec sporotrichides vient d'être présenté. Des cultures satisfaisantes ont été obtenues seulement après inoculation des animaux. La cure à l'Organidin, une forme organique de l'iodine, à été proposée quand survient une sérieuse sensibilité à KI.On insist sur le fait que la sporotrichose à des nombreuses formes cliniques.

     

Chronic pulmonary sporotrichosis

We report a case of chronic human pulmonary sporotrichosis which was not associated with superficial manifestations involving the skin or lymph nodes. As it is difficult to verify an etiology, it is possible that some undiagnosed pulmonary granulomas could represent pulmonary sporotrichosis of the chronic type. These may have been treated for other diseases, such as a mycobacterial infection as in this case. Since the immunodiffusion test is a simple procedure and is almost always positive in chronic disease, its greater utilization should help in defining this disease more frequently. The skin test, agglutination test and culture are variably useful as documented in the literature.Sporotrichosis is a common mycotic infection which is usually confined to the skin and superficial lymph nodes. In disseminated disease, the lung is very rarely involved. Pulmonary sporotrichosis then represents a second form of primary infection though less common than the lymphocutaneous disease. Scott et al. (18) reported two cases of pulmonary sporotrichosis. Ridgeway et al. (15) at about the same time reviewed the previously reported cases up to 1962 and concluded that 12 of the cases could be accepted as sporotrichosis and added two cases of their own. Subsequently, Siegrist & Ferrington (20) and Trevathan & Phillips (23) each reported a case. Presently, approximately 50 cases have been reported in the United States (1–10, 12, 13, 16). Chronic and acute involvement have been delineated by Ridgeway et al. (15) It is believed that the following case represents one of primary pulmonary sporotrichosis of the chronic type without manifest involvement of other organs.     

Immune Response Against Sporothrix schenckii in TLR-4-Deficient Mice

For many fungal diseases, macrophages are the major cell population implicated in host protection, primarily by their ability to eliminate the invading fungal pathogen through phagocytosis. In sporotrichosis, this remains true, because of macrophages’ ability to recognize Sporothrix schenckii through specific receptors for some of the fungus’ cellular surface constituents. Further confirmation for macrophages’ pivotal role in fungal diseases came with the identification of toll-like receptors, and the subsequent numerous associations found between TLR-4 deficiency and host susceptibility to diverse fungal pathogens. Involvement of TLR-4 in immune response against sporotrichosis has been conducted to investigate how TLR-4 signaling could affect inflammatory response development through evaluation of H2O2 production and IL-1β, IL-6 and TGF-β release during the course of S. schenckii infection on TLR-4-deficient mice. The results showed that macrophages are largely dependent on TLR-4 for inflammatory activation and that in the absence of TLR-4 signaling, increased TGF-β release may be one of the contributing factors for the abrogated inflammatory activation of peritoneal exudate cells during mice sporotrichosis.     

Variation in Genotype and Higher Virulence of a Strain of <Emphasis Type="Italic">Sporothrix schenckii</Emphasis> Causing Disseminated Cutaneous Sporotrichosis

Sporotrichosis is usually a localized, lymphocutaneous disease, but its disseminated type was rarely reported. The main objective of this study was to identify specific DNA sequence variation and virulence of a strain of Sporothrix schenckii isolated from the lesion of disseminated cutaneous sporotrichosis. We confirmed this strain to be S. schenckii by^® tubulin and chitin synthase gene sequence analysis in addition to the routine mycological and partial ITS and NTS sequencing. We found a 10-bp deletion in the ribosomal NTS region of this strain, in reference to the sequence of control strains isolated from fixed cutaneous sporotrichosis. After inoculated into immunosuppressed mice, this strain caused more extensive system involvement and showed stronger virulence than the control strain isolated from a fixed cutaneous sporotrichosis. Our study thus suggests that different clinical manifestation of sporotrichosis may be associated with variation in genotype and virulence of the strain, independent of effects due to the immune status of the host.     

难辨认孢子丝菌病临床分析

探讨孢子丝菌病不典型的临床表现.孢子丝菌病是皮肤科常见的一种深部真菌病,典型的皮肤科特征是慢性肉芽肿、溃疡性损害、炎性结节等.而部分孢子丝菌病的患者由于各种各样的原因导致皮损丧失特征性临床表现,给皮肤科医生尤其是临床经验尚不丰富的年轻医师于该病的诊断带来了一定的困难.本文所讨论的8例患者均为就诊于我科门诊并已确诊为孢子丝菌病病例,皮损与典型的孢子丝菌病皮损表现不相符,希望皮肤科医师在以后的临床工作中遇到一些不典型皮损但是综合患者病史、生活环境、诊治过程等情况可考虑到此种疾病,及时给予明确的诊断,使患者能够得到及时、有效的治疗.     

Sporotrichosis in Rio de Janeiro,Brazil: Sporothrix brasiliensis Is Associated with Atypical Clinical Presentations

Background

There have been several recent changes in the taxonomy of Sporothrix schenckii as well as new observations regarding the clinical aspects of sporotrichosis. In this study, we determined the identification of the Sporothrix species associated with both classic and unusual clinical aspects of sporotrichosis observed in the endemic area of sporotrichosis in Rio de Janeiro, Brazil.

Methodology/Principal Findings

To verify whether S. brasiliensis is associated with clinical manifestations of sporotrichosis, a cross-sectional study was performed in which Sporothrix isolates from 50 patients with different clinical manifestations were analyzed and their isolates were studied by phenotypic and genotypic methods. Data from these patients revealed a distinct clinical picture and therapeutic response in infections caused by Sporothrix brasiliensis (n = 45) compared to patients with S. schenckii sensu stricto (n = 5). S. brasiliensis was associated with disseminated cutaneous infection without underlying disease, hypersensitivity reactions, and mucosal infection, whereas patients with S. schenckii presented with less severe and more often localized disease, similar to the majority of previously described sporotrichosis cases. Interestingly, S. brasiliensis-infected patients overall required shorter durations of itraconazole (median 16 weeks) compared to the individuals with S. schenckii (median 24 weeks).

Conclusions/Significance

These findings suggest that Sporothrix species are linked to different clinical manifestations of sporotrichosis and that S. brasiliensis is effectively treated with oral itraconazole.     

Cytokine responsiveness in germfree and conventional NMRI mice

We have investigated the proliferative response of thymocytes from different mouse strains to cytokines in vitro. Interleukin 2 (IL-2), IL-4 and IL-7 induced proliferation of thymocytes from NMRI/KI (a locally bred NMRI mouse strain), NMRI/H ('traditional' NMRI mice), C3H/HeJ and C3H/HeN mice. NMRI/KI thymocytes showed the most prominent proliferation in response to IL-1 alpha and IL-1 beta. IL-3, IL-5, IL-6, IL-8, IL-10, tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), inhibin and lipopolysaccharide (LPS) induced no thymocyte proliferation. Germfree NMRI/KI mouse thymocytes showed a significantly lower proliferation in response to IL-1 alpha and IL-1 beta than conventional mice. Rat tissues, previously shown to contain lymphocyte activating factors (LAFs), were also tested. Skin, tongue, esophagus, proventricular stomach, testis and placenta were all positive in the LAF assay utilizing NMRI/KI thymocytes, whereas none of the tissue extracts could induce proliferation in NMRI/H thymocytes. The higher cytokine responsiveness in conventional mice compared with germfree might suggest that exposure to microflora induces a higher state of activation of the immune system. The LAF assay, utilizing NMRI/KI thymocytes, is a highly sensitive IL-1 bioassay with a detection level of 1 pg/ml for IL-1 beta and 2 pg/ml for IL-1 alpha. The specificity of the assay is increased by utilizing NMRI/H mice to exclude the presence of IL-2, IL-4 and IL-7.     

Proteomics-Based Characterization of the Humoral Immune Response in Sporotrichosis: Toward Discovery of Potential Diagnostic and Vaccine Antigens

Background

Sporothrix schenckii and associated species are agents of human and animal sporotrichosis that cause large sapronoses and zoonoses worldwide. Epidemiological surveillance has highlighted an overwhelming occurrence of the highly pathogenic fungus Sporothrix brasiliensis during feline outbreaks, leading to massive transmissions to humans. Early diagnosis of feline sporotrichosis by demonstrating the presence of a surrogate marker of infection can have a key role for selecting appropriate disease control measures and minimizing zoonotic transmission to humans.

Methodology

We explored the presence and diversity of serum antibodies (IgG) specific against Sporothrix antigens in cats with sporotrichosis and evaluated the utility of these antibodies for serodiagnosis. Antigen profiling included protein extracts from the closest known relatives S. brasiliensis and S. schenckii. Enzyme-linked immunosorbent assays and immunoblotting enabled us to characterize the major antigens of feline sporotrichosis from sera from cats with sporotrichosis (n = 49), healthy cats (n = 19), and cats with other diseases (n = 20).

Principal Findings

Enzyme-linked immunosorbent assay-based quantitation of anti-Sporothrix IgG exhibited high sensitivity and specificity in cats with sporotrichosis (area under the curve, 1.0; 95% confidence interval, 0.94–1; P<0.0001) versus controls. The two sets of Sporothrix antigens were remarkably cross-reactive, supporting the hypothesis that antigenic epitopes may be conserved among closely related agents. One-dimensional immunoblotting indicated that 3-carboxymuconate cyclase (a 60-kDa protein in S. brasiliensis and a 70-kDa protein in S. schenckii) is the immunodominant antigen in feline sporotrichosis. Two-dimensional immunoblotting revealed six IgG-reactive isoforms of gp60 in the S. brasiliensis proteome, similar to the humoral response found in human sporotrichosis.

Conclusions

A convergent IgG-response in various hosts (mice, cats, and humans) has important implications for our understanding of the coevolution of Sporothrix and its warm-blooded hosts. We propose that 3-carboxymuconate cyclase has potential for the serological diagnosis of sporotrichosis and as target for the development of an effective multi-species vaccine against sporotrichosis in animals and humans.     

Comparative Evaluation of Five Serological Methods for the Diagnosis of Sporotrichosis

The diagnosis of sporotrichosis can be time consuming. Serological procedures could facilitate the rapid and accurate diagnosis of this disease. A slide latex agglutination (SLA) test for sporotrichosis was developed and compared with the tube agglutination (TA), complement fixation (CF), and immunodiffusion (ID) tests in the serological study of 80 proven human cases of sporotrichosis representing the cutaneous, subcutaneous, and extracutaneous forms of the disease. In addition, the indirect fluorescent antibody (IFA) technique was applied to 61 case sera. In the SLA test, latex particles sensitized with culture filtrate antigens from the yeast form of Sporothrix schenckii (B 959) detected 94% of the cases, as compared to 96% of the cases detected by the TA test, 68% by the CF test, and 56% by the ID test. The IFA test detected 90% of the 61 cases. The SLA and ID tests were specific, showing no reactions with sera from 86 persons with no disease or with diseases other than sporotrichosis. Because of its sensitivity, specificity, ease of performance, and ability to provide results in 5 min, the SLA test is highly recommended for routine use in the clinical laboratory.     

The Predominance of Alternatively Activated Macrophages Following Challenge with Cell Wall Peptide-Polysaccharide After Prior Infection with Sporothrix schenckii

Sporotrichosis is a subcutaneous mycosis that is caused by the dimorphic fungus Sporothrix schenckii. This disease generally occurs within the skin and subcutaneous tissues, causing lesions that can spread through adjacent lymphatic vessels and sometimes leading to systemic diseases in immunocompromised patients. Macrophages are crucial for proper immune responses against a variety of pathogens. Furthermore, macrophages can play different roles in response to different microorganisms and forms of activation, and they can be divided into “classic” or “alternatively” activated populations, as also known as M1 and M2 macrophages. M1 cells can lead to tissue injury and contribute to pathogenesis, whereas M2 cells promote angiogenesis, tissue remodeling, and repair. The aim of this study was to investigate the roles of M1 and M2 macrophages in a sporotrichosis model. Toward this end, we performed phenotyping of peritoneal exudate cells and evaluated the concomitant production of several immunomediators, including IL-12, IL-10, TGF-β, nitric oxide, and arginase-I activity, which were stimulated ex vivo with cell wall peptide-polysaccharide. Our results showed the predominance of the M2 macrophage population, indicated by peaks of arginase-I activity as well as IL-10 and TGF-β production during the 6th and 8th weeks after infection. These results were consistent with cellular phenotyping that revealed increases in CD206-positive cells over this period. This is the first report of the participation of M2 macrophages in sporotrichosis infections.     

Diagnosis and Treatment of Lymphocutaneous Sporotrichosis: What Are the Options?

Lymphocutaneous sporotrichosis is the most common clinical form of cutaneous sporotrichosis. Caused by a complex of dimorphic fungi called Sporothrix schenckii complex, it is an occupational disease, present especially in tropical and subtropical areas, and has been reported in all continents. Diagnosis is established by isolation of the causative agent. Therapy of choice for lymphocutaneous sporotrichosis is itraconazole, and in developing countries, potassium iodide solution. In general, the lymphocutaneous form is considered a mild benign form of the disease, and the majority of cases respond well to treatment in about 3–4 months of therapy. In this paper, we have made a general review of the disease, especially of the epidemiology, clinical features and diagnosis of the lymphocutaneous form, as well as a brief analysis of the advantages and disadvantages of diverse treatments.     

Store-operated Ca2+ entry through ORAI1 is critical for T cell-mediated autoimmunity and allograft rejection

ORAI1 is the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel, which is responsible for store-operated Ca(2+) entry in lymphocytes. A role for ORAI1 in T cell function in vivo has been inferred from in vitro studies of T cells from human immunodeficient patients with mutations in ORAI1 and Orai1(-/-) mice, but a detailed analysis of T cell-mediated immune responses in vivo in mice lacking functional ORAI1 has been missing. We therefore generated Orai1 knock-in mice (Orai1(KI/KI)) expressing a nonfunctional ORAI1-R93W protein. Homozygosity for the equivalent ORAI1-R91W mutation abolishes CRAC channel function in human T cells resulting in severe immunodeficiency. Homozygous Orai1(KI/KI) mice die neonatally, but Orai1(KI/KI) fetal liver chimeric mice are viable and show normal lymphocyte development. T and B cells from Orai1(KI/KI) mice display severely impaired store-operated Ca(2+) entry and CRAC channel function resulting in a strongly reduced expression of several key cytokines including IL-2, IL-4, IL-17, IFN-γ, and TNF-α in CD4(+) and CD8(+) T cells. Cell-mediated immune responses in vivo that depend on Th1, Th2, and Th17 cell function were severely attenuated in ORAI1-deficient mice. Orai1(KI/KI) mice lacked detectable contact hypersensitivity responses and tolerated skin allografts significantly longer than wild-type mice. In addition, T cells from Orai1(KI/KI) mice failed to induce colitis in an adoptive transfer model of inflammatory bowel disease. These findings reaffirm the critical role of ORAI1 for T cell function and provide important insights into the in vivo functions of CRAC channels for T cell-mediated immunity.     

Disseminated sporotrichosis with immune reconstitution inflammatory syndrome in an HIV patient: Case report and review of the literature

BackgroundSporotrichosis has been occurring as outbreaks in Brazil, reaching epidemic levels in some regions. Zoonotic transmission is the main route to acquire Sporothrix.Case reportWe describe a case of disseminated sporotrichosis caused by Sporothrix brasiliensis in an HIV/AIDS patient, with the presentation of immune reconstitution inflammatory syndrome (IRIS).ConclusionsThis case reinforces that sporotrichosis should always be suspected in patients with IRIS from endemic regions, even in patients without the typical cutaneous lesions of this mycosis.     

T15-idiotype-negative B cells dominate the phosphocholine binding cells in the preimmune repertoire of T15i knockin mice.

T15i knockin (KI) mice express a H chain that is encoded by a rearranged T15 VDJ transgene which has been inserted into the J(H) region of chromosome 12. This T15H chain combines with a kappa22-33 L chain to produce a T15-Id+ Ab having specificity for phosphocholine (PC). Inasmuch as T15-Id+ Abs dominate the primary immune response to PC in normal mice, it was surprising to find that 80% of the PC-dextran-binding B cells in unimmunized homozygous T15i KI mice were T15-Id-. Analysis of L chains expressed in these T15-Id-, PC-specific B cells revealed that two L chains, kappa8-28 and kappa19-15, were expressed in this population. The V(kappa) region of these L chains was recombined to J(kappa)5, which is typical of L chains present in PC-specific Abs. When T15i KI mice were immunized with PC Ag, T15-Id+ B cells expanded 6-fold and differentiated into Ab-secreting cells. There was no indication that the T15-Id- B cells either proliferated or differentiated into Ab-secreting cells following immunization. Thus, T15-Id- B cells dominate the PC-binding population, but they fail to compete with T15-Id+ B cells during a functional immune response. Structural analysis of T15H:kappa8-28L and T15H:kappa19-15L Abs revealed L chain differences from the kappa22-33 L chain which could account for the lower affinity and/or avidity of these Abs for PC or PC carrier compared with the T15-Id+ T15H:kappa22-33L Ab.