Immunohistochemistry for the diagnosis of aspergillosis in Turkey poults.

From each of two flocks (A and B) of poults comprising 14,100 females and 11,300 males, respectively, 15 poults were examined pathologically. Poults of flock A had signs of neurological disturbances whereas birds from flock B showed respiratory symptoms. Gross lesions were observed only in two poults from flock A in which minute circular areas of cerebral malaria were seen. Histopathologically, the brain lesions contained fungal elements, and so did some of the pulmonary granulomas detected in three and six poults out of four and six birds examined from flock A and B, respectively. Mycological cultivation was attempted from the brains and lungs of five poults from flock A. However, only from the brain of a single bird a fungus, identified as Aspergillus fumigatus, was grown. Immunohistochemistry was applied because the histomorphology of fungal elements within some lesions did not offer any characteristics allowing an assessment of the identity of the infective fungi. Moreover, as fungi could not be detected within all lesions, immunohistochemistry accomplished the screening of tissues. For immunostaining of tissues a panel of monoclonal and polyclonal antibodies identifying agents of aspergillosis, candidosis, fusariosis, scedosporiosis, and zygomycosis, was used. Due to a strong and uniform reactivity of all fungal elements with immunoreagents to Aspergillus spp. an unequivocal diagnosis of aspergillosis was established in all mycotic lesions. Apart from the establishment of an aetiological diagnosis, the application of immunohistochemistry also disclosed fungal fragments in granulomas which could not be identified with conventional histochemical stains.     

The role of neutrophils in the development and outcome of zygomycosis in haematological patients

Zygomycosis constitutes the third leading cause of invasive fungal infections following aspergillosis and candidosis. Patients with haematologic malignancies or haematopoietic stem cell transplantation are particularly susceptible to zygomycosis. Neutropenia represents the most important pathogenic mechanism influencing the onset and outcome of zygomycosis. Neutrophils cause a lesion of the fungal wall with subsequent destruction by macrophages. They also enhance the activity of antifungal drugs against Zygomycetes. Strategies that aim to increase neutrophil count and function, such as granulocyte colony stimulating factor and granulocyte transfusion, could play an important role in the management of this life-threatening infectious complication.     

New developments in the diagnosis of opportunistic fungal infection

This review considers recent developments in the diagnosis of aspergillosis, candidosis and cryptococcosis and discusses the prospects for routine application of a number of novel methods. The introduction of lysis-centrifugation and radiometric methods for blood culture has improved the diagnosis of deep candidosis, but the value of these methods for the diagnosis of aspergillosis has not yet been determined. Recent developments in serological diagnosis have included the evaluation of newly discovered antigens of Candida albicans in an attempt to distinguish colonization from significant infection. Antigen detection, an established method for the diagnosis of cryptococcosis, has also been evaluated and appears promising for the diagnosis of aspergillosis and candidosis. Another promising approach has been the use of gas-liquid chromatography to detect fungal metabolites in serum and other host fluids.     

Diagnosis of Disseminated Zygomycosis Using a Polymerase Chain Reaction Assay

Invasive pulmonary zygomycosis is an uncommon opportunistic infection in patients with haematological malignancies. Clinical manifestations are indistinguishable from the more frequent invasive aspergillosis. Standard diagnostic methods like culture and microscopy from respiratory secretions have a low diagnostic sensitivity. A case in which proven invasive pulmonary zygomycosis was confirmed using a panfungal polymerase chain reaction assay in blood is presented. Since zygomycosis requires more aggressive treatment than aspergillosis (high-dose amphotericin B and surgical intervention), the polymerase chain reaction assay may improve the outcome of these often fatal infections by guiding the therapeutic approach through an early, noninvasive diagnosis. Electronic Publication     

Value of detection of antibodies toCandida albicans germ tube in the diagnosis of systemic candidosis

To test the value of detection of anti-Candida albicans germ tube antibodies by indirect immunofluorescence assay in the diagnosis of systemic candidosis, a retrospective study was done using 126 sera from 27 patients with presumptive systemic candidosis (13 immunocompromised), 165 sera from 45 patients with aspergillosis (29 immunocompromised), 35 sera from eight patients with cryptococcosis (6 immunocompromised), and 101 sera from 101 blood donors. While 21 of 27 patients with systemic candidosis (77.8%) had anti-germ tube antibodies, these antibodies were absent in all patients with cryptococcosis and in all blood donors. They were however detected in 5 of 45 patients with aspergillosis (11.1%). Ten of 13 (76.9%) immunocompromised patients with candidosis had anti-germ tube antibodies; similar results were obtained in immunocompetent patients with candidosis (78.6%). The specificity was 96.8%, indicating a high degree of discrimination was possible between systemic candidosis and other invasive mycoses in the patients studied. Anti-germ tube responses did not appear to be significantly reduced in immunocompromised patients.     

Cutaneous manifestations of systemic mycoses]

The systemic mycoses are increasing in importance as opportunistic infections. Cutaneous lesions resulting from systemic mycoses may first alert clinicians to the presence of a life-threatening disorder, or even the presence of an unsuspected immunodeficiency state. Skin involvement is generally uncommon in disseminated aspergillosis, zygomycosis but is more common in systemic candidiasis (candidemia) and cryptococcosis. The blanket terms, hyalohyphomycosis and phaeohyphomycosis, cover the infections caused by diverse fungal opportunists. A variety of manifestations of skin lesions of the systemic mycoses are reviewed. These specific and/or non-specific lesions require early recognition, diagnosis, and aggressive antifungal treatment.     

Epidemiology of visceral mycoses in autopsy cases in Japan: comparison of the data from 1989, 1993, 1997, 2001, 2005 and 2007 in Annual of Pathological Autopsy Cases in Japan

The data on visceral mycoses reported in the " Annual of Pathological Autopsy Cases in Japan " were analyzed epidemiologically every four years from 1989 to 2005, and in 2007. The frequency rates of visceral mycoses dropped sharply between 1989 (4.5%) and 1994 (3.2%), but by 2001 had risen again and have remained (4.4-4.6%) generally stable since then. The predominant causative agents were Candida and Aspergillus. Although the rate of candidosis showed a gradual decrease, the rate of aspergillosis showed an increase by degrees. Furthermore, the rate of aspergillosis exceeded that of candidosis in 1994, and the difference in the rates between the two conditions apparently further increased until 2001. After 2005, however no changes in this difference were observed. For complicated infections, the incidence of coinfection with Aspergillus and Candida showed a decreasing, and that with Aspergillus and Zygomycetes showed an increasing tendency. Severe infections with Zygomycetes showed a clear increase from 57.4% in 1989 to 88.9% in 2007. Comparing underlying diseases with mycoses in 1989 and 2007, leukemia (including myelodysplastic syndrome) decreased from 26.1% to 18.8% and bacterial infections (including interstitial pneumonia) increased from 11.1% to 22.1%. By age, the highest frequency rate of mycoses was observed in the range of 60-79 years, and the frequency rate of exogenous fungal infections such as aspergillosis, cryptococcosis, zygomycosis and trichosporonosis showed an increasing trend in the less than one-year old group.     

Clinical evaluation of diagnostic methods using plasma and/or serum for three mycoses: aspergillosis, candidosis, and pneumocystosis.

Clinical evaluation was retrospectively made of the results of serological diagnostic methods using plasma and/or sera of patients for the diagnosis of aspergillosis, candidosis, and pneumocystosis. Specimens were drawn from 8 patients with invasive aspergillosis, 3 with aspergilloma, 9 with candidosis, 4 with pneumocystosis, and 15 with no fungal infections. In invasive aspergillosis, the sensitivities of the (1-3)-beta-D-glucan measurement test using chromogenic and turbidimetric methods were 78.6% and 82.1%, with specificities of 75% and 87.5%, respectively. The sensitivity of the Pastorex Aspergillus test for invasive aspergillosis was 16.7%, with a specificity of 92.3%. In candidosis, the sensitivities of the (1-3)-bata-D-glucan test using the above two methods were 84.2% and 100%, with specificities of 75% and 87.5%, respectively. The sensitivity of the CAND-TEC test and the Pastorex Candida test for candidosis were 68.8% and 16.7%, with specificities of 57.1% and 100%, respectively. These results indicate that the (1-3)-bata-D-glucan measurement methods are more reliable in clinical application than the other antigen detection methods, but they still lack efficiency in differentiating fungal infections such as aspergillosis, candidosis and pneumocystosis. For a more exact diagnosis of systemic fungal infections, detailed studies on the clinical symptoms are considered essential.     

The emergence of candidosis. The dominant postmortem cerebral mycosis

Comparable human postmortem surveys in central Kentucky and southern Florida have demonstrated an altered pattern of cerebral mycoses due primarily to therapeutic manipulations. From both states 8,975 complete autopsies yielded 39 patients with histologically verified cerebral mycoses. The most common infection was candidosis (49%), characterized by intraparenchymal microabscesses without significant leptomeningitis in hospitalized patients compromised by antibiotic therapy for infection with gram-negative organisms. The remaining 20 patients with noncandidal cerebral mycoses included 9 (23%) with cryptococcosis, 5 (13%) with zygomycosis, 2 (5%) with aspergillosis, 2 (5%) with histoplasmosis, 1 (2.5%) with blastomycosis and 1 (2.5%) with curvulariosis. These compromised patients had leptomeningitis when infected with yeasts and cerebral infarcts with cerebritis when infected with hyphal fungi. In contrast to human cerebral candidosis, the non-candidal cerebral mycoses precipitated the patient's terminal hospitalization. These infections seemed to be contracted outside the hospital. Therapy for gram-negative bacterial infections has enabled Candida species to overtake Cryptococcus neoformans as the most common cause of postmortem cerebral mycosis.     

PCR based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue

BACKGROUND: Invasive fungal infections are often diagnosed by histopathology without identification of the causative fungi, which show significantly different antifungal susceptibilities. AIMS: To establish and evaluate a system of two seminested polymerase chain reaction (PCR) assays to identify and discriminate between agents of aspergillosis and mucormycosis in paraffin wax embedded tissue samples. METHODS: DNA of 52 blinded samples from five different centres was extracted and used as a template in two PCR assays targeting the mitochondrial aspergillosis DNA and the 18S ribosomal DNA of zygomycetes. RESULTS: Specific fungal DNA was identified in 27 of 44 samples in accordance with a histopathological diagnosis of zygomycosis or aspergillosis, respectively. Aspergillus fumigatus DNA was amplified from one specimen of zygomycosis (diagnosed by histopathology). In four of 16 PCR negative samples no human DNA was amplified, possibly as a result of the destruction of DNA before paraffin wax embedding. In addition, eight samples from clinically suspected fungal infections (without histopathological proof) were examined. The two PCR assays detected a concomitant infection with Absidia corymbifera and A fumigatus in one, and infections with Rhizopus arrhizus and A fumigatus in another two cases. CONCLUSIONS: The two seminested PCR assays described here can support a histopathological diagnosis of mucormycosis or aspergillosis, and can identify the infective agent, thereby optimising antifungal treatment.     

Serological tests in the diagnosis of pulmonary aspergillosis

Sera from 44 patients with clinically suspected pulmonary aspergillosis (mainly of an allergic type) were examined for antibodies to Aspergillus fumigatus using an enzyme-linked immunosorbent assay (ELISA) and counter-immunoelectrophoresis (CIE). Of the sera, 15 were considered to contain Aspergillus antibodies using ELISA; 11 of these also contained precipitins by CIE. In no instance was a CIE-positive serum negative by ELISA. The serum of 1 patient with autopsy-verified invasive pulmonary aspergillosis was negative in both tests. Protein-enriched antigens derived by ammonium sulphate precipitation of crude hyphal homogenates seemed of most use in ELISAs. In addition, 4 of 8 sera obtained from patients with possibly invasive candidosis also revealed significant levels of Aspergillus antibody by ELISA (but not CIE). All of the 8 sera contained readily detectable Candida precipitins by CIE. Problems of potential cross-reactivity obviously need careful consideration with ELISAs. Our results suggest that ELISA procedures under appropriately controlled conditions are more sensitive than CIE for detecting Aspergillus antibodies. However it seems that some patients with invasive aspergillosis will be antibody-negative even with sensitive tests such as ELISA.     

COMPUTERIZED SCENE SEGMENTATION FOR THE DISCRIMINATION OF ARCHITECTURAL FEATURES IN DUCTAL PROLIFERATIVE LESIONS OF THE BREAST

The distinction between ductal hyperplasia (DH) and ductal carcinoma in situ (DCIS) still remains a problem in the histological diagnosis of non-invasive breast lesions. In this study, a method was developed for the automatic segmentation and quantitative analysis of breast ducts using knowledge-guided machine vision. This permitted duct profiles and intraduct lumina to be identified and their shape, size, and number computed. These were used to derive measures of duct cribriformity and architectural complexity which were examined as an objective tool in the characterization of duct pattern in proliferative lesions. A total of 215 images of ducts were digitally captured from 22 cases of DCIS and 21 cases of DH diagnosed independently by two pathologists. The cribriformity index proved to be a useful measure of duct architecture, showing a monotonic increase with increasing duct complexity. The number of lumina also increased with increasing overgrowth of ductal epithelium until the duct was filled. Discriminant analysis of the duct characteristics for benign and malignant groups selected the lumen area/duct area ratio and the duct area as significant discriminatory variables and they were combined into a discriminant function. Of the lumen features, the mean area of the lumen and the polar average (mean of the distribution of the number of events with an increasing spiral from the centre of the duct) were combined into a second discriminant function. Plotting cases against these two functions provided good separation of DH and DCIS groups, with correct classification estimated on the training sample as being over 80 per cent. With an increasing incidence of complex proliferative lesions arising from mammography, the ability to diagnose these lesions correctly is more important than ever. The use of expert system-guided machine vision facilitates the quantitative evaluation of breast duct architecture; along with established histological and cytological criteria, it is hoped that this will lead to a more objective means of diagnosis and disease classification. © 1997 by John Wiley & Sons, Ltd.     

The Budd-Chiari syndrome caused by a zygomycete. A new pathogenesis of hepatic vein thrombosis

The clinical history of a 60-year-old woman suffering from chronic lymphocytic leukemia with sudden deterioration and death is reported. The postmortem macroscopic and microscopic findings included pulmonary aspergillosis and pulmonary zygomycosis (mucormycosis). Hematogenous dissemination of the zygomycete causing cardiac zygomycosis, cerebral infarcts due to vascular occlusion by hyphae, and thrombosis of the major hepatic veins (Budd-Chiari syndrome) with submassive necrosis of the right liver lobe were also observed. To our knowledge, this is the second report dealing with occlusion of the hepatic veins caused by a fungus and the first study reporting the Budd-Chiari syndrome due to a mold of the subclass Zygomycetes.     

Zygomycosis: two case reports and review of reported cases in the literature in Japan]

This article reports two cases of zygomycosis and analyzes the zygomycosis cases reported in the literature in Japan. Case 1 was a 43-year-old male with malignant lymphoma who presented complications of pneumonia and cerebral bleeding, leading to his death. Autopsy findings showed pulmonary lesions were due to zygomycosis. Cerebral lesion was presumed to be due to zygomycosis without pathological examination. Case 2 was a 52-year-old male with acute lymphocytic leukemia from whom 4 sputum cultures were taken that were positive for Cunninghamella elegans. Combination therapy of itraconazole and amphotericin B (AMPH) was begun, and AMPH was changed to liposomal amphotericin B. During the neutropenic period after receiving premedication for a peripheral blood stem cell transplantation performed for his underlying disease, high fever was recognized and Staphylococcus epidermidis was isolated from the blood culture. Despite the change in antibiotics administered, pneumonia also developed as a complication, causing his death. Two hundred four cases of zygomycosis have been reported in the literature in Japan: 55 cases were rhinocerebral zygomycosis, including 29 cases with no underlying disease. A premortem diagnosis was made in 34 cases by pathological findings of operation materials or drainage samples, and 24 cases were postmortem. Pulmonary, disseminated, cardiovascular, gastrointestinal and thyroidal zygomycoses were found in 144 cases, including 66 cases with leukemia. A premortem diagnosis was made in 39 cases and 120 cases were postmortem. Prognosis of rhinocerebral type was better in operated or drainage cases, and for resected cases in all other types. Five cases with allergic zygomycosis were all alive. There were only 14 cases in which isolated fungi were identified (Cunninghamella spp. from 5 cases, Mucor spp. from 2, Rhizomucor spp. from 2, and Rhizopus spp. from 5).     

Vesicular thick-walled swollen hyphae in pulmonary zygomycosis

An autopsy case of pulmonary zygomycosis in a patient with rheumatoid arthritis on immunosuppressive therapy is presented herein. There was a pulmonary cavitated infarct caused by mycotic thrombosis. Thin-walled narrow hyphae and vesicular thick-walled swollen hyphae were found on the pleural surface and in the necrotic tissue at the periphery of the cavity. Findings of such shaped fungal elements may cause erroneous histopathological diagnosis because pauciseptate broad thin-walled hyphae are usually the only detectable fungal elements in zygomycosis tissue. Although immunohistochemistry confirmed these unusual elements to be zygomycetous in the present case, it is important for the differential diagnosis to be aware that zygomycetes can form thin narrow hyphae and vesicular thick-walled swollen hyphae.     

人胚胎结肠肠神经系统发育的观察

目的　研究人胚胎结肠肠神经系统的发育过程 ,为进一步研究先天性巨结肠的发病机制提供参考。　方法　采用一抗为蛋白基因产物 (protein gene product9.5 ,PGP9.5 )和 S- 10 0蛋白抗体的免疫组织化学 PAP法 ,显示结肠肠神经系统中的神经元和神经胶质。　结果　人胚胎结肠肠神经系统发育有明显的阶段性。在胚胎发育早期 (胎龄 2～ 3月 ) ,肠管壁发育差 ,以后出现菲薄的平滑肌层和低平的肠粘膜 ,此期偶在原始肌间神经丛位置见S- 10 0蛋白免疫反应性神经 ;至发育中期 (胎龄 4～ 5月 ) ,肠壁分化出 4层结构 ,出现相当发达的绒毛 ,肌间神经丛中细胞明显增多 ,呈弥散分布于整个肌层间并逐渐迁移到粘膜下层和粘膜层 ,由初级和次级突起构成复杂的神经网络 ;至晚期 (6～ 9月 ) ,肠壁各层均增厚 ,肌间神经丛成簇分布 ,神经纤维构成的网络出现更为细小的 3级突起 ,粘膜下神经丛分化形成浅丛和深丛。　结论　结肠神经系的发育具有明显的阶段性。发育早期神经开始在肠壁肌间丛位置出现 ,发育中期神经成分在肠壁各层中出现并发育增生 ,晚期肠壁各层神经已分化和成熟 ,而在不同的发育阶段 ,原始病因可导致临床表现不一的先天性巨结肠症     

Incidence of zygomycosis in transplant recipients

Recently, a remarkable increase in the incidence of zygomycosis has been reported from institutions in the USA and Europe. The use of voriconazole for the treatment of aspergillosis and, less frequently, the use of echinocandins as empirical treatment for invasive fungal infections are thought to be responsible for the increase. In addition, an increased incidence of this infection has been observed in transplant recipients, including both haematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) patients. There are no global surveys on the prevalence or incidence of zygomycosis, but data from individual institutions and countries show that zygomycosis is an emerging infection. The increased incidence of zygomycosis most probably reflects a greater frequency of predisposing factors, such as higher numbers of patients undergoing HSCT and immunosuppressive chemotherapy. In addition, the emergence of rare pathogens as a result of the rise in the use of antifungal therapy against common species can be postulated. Further, the availability of antifungal agents with activity profiles that are more specific for selected fungi increases the necessity of identifying pathogenic fungi; the frequency of Zygomycetes infections may have been underestimated until now because therapeutic decisions did not depend on the precise identification of pathogenic fungi.     

Deposition of calcium salts in a case of pulmonary zygomycosis: histopathologic and chemical findings

We report a case of pulmonary zygomycosis associated with unusual deposition of calcium salt crystals. The patient was a 75-year-old female who had onset of cough and shortness of breath. She was treated for community-acquired pneumonia but died despite intensive therapy. Postmortem examination revealed diffuse alveolar damage and multifocal necrotizing pneumonia associated with herpes simplex infection and invasive zygomycosis. Birefringent particles were seen associated with fungal elements in the lung parenchyma, within bronchial cartilage, and in blood vessel walls. By infrared spectroscopy, the birefringent particles in the pulmonary parenchyma and within bronchial cartilage had spectral characteristics of calcium oxalate dihydrate and calcium oxalate monohydrate, respectively. The birefringent crystals within vascular walls were identified as calcium carbonate. This case documents the chemical composition and location of 3 different calcium salt crystals in pulmonary zygomycosis. It also shows that among pulmonary fungal infections, calcium oxalate deposition is not restricted to aspergillosis.     

Anti-Candida activity of azoles

The in vitro activity of the broad-spectrum antifungals miconazole, ketoconazole and itraconazole was evaluated by the decimal dilution method in liquid media, for respectively 2511, 1536 and 1859 strains of yeasts belonging to 31 species. Sabouraud broth was used for miconazole and brain heart infusion broth for ketoconazole and itraconazole. These three azoles proved to be potent anti-yeast compounds. Activity by oral treatment of miconazole, ketoconazole and itraconazole was compared in gastro-intestinal candidosis of the guinea-pig, in vaginal candidosis of the rat and in systemic candidosis of the guinea-pig. Ketoconazole and itraconazole are more efficacious than miconazole in gastro-intestinal candidosis. Miconazole showed marginal activity in the other two experimental models: the spectrum of activity of miconazole was a lead for research which resulted in the synthesis and selection of ketoconazole and itraconazole. Ketoconazole was highly active in these experimental models. Itraconazole cured the animals at distinctly lower doses. No side-effects due to these azoles were observed during these experiments. A proposal is made to classify the broad-spectrum azoles in three classes: compounds for topical use (e.g. miconazole), compounds with oral activity without activity in aspergillosis (e.g. ketoconazole), compounds with oral activity including activity in aspergillosis (e.g. itraconazole).     

Changing epidemiology of an emerging infection: zygomycosis

Aspergillosis and candidosis remain the most prevalent opportunistic fungal infections in immunocompromised patients, but diseases caused by the Zygomycetes have become of increasing importance. Exposure to antimycotic drugs with no activity against zygomycetes may be a new risk factor and an explanation for the increasing incidence of zygomycosis. The latter infection occurs only rarely in immunocompetent hosts, but in recent years Apophysomyces elegans has been described in many subtropical countries as an emerging pathogen causing mostly cutaneous infections after traumatic inoculation.