Blastomyces dermatitidis in bats: first report of its isolation from the liver of Rhinopoma hardwickei hardwickei Gray

Blastomyces dermatitidis is reported for the first time from the liver of Rhinopoma hardwickei hardwickei Gray (the 'lesser rat-tailed bat'); it was cultured from one of 46 samples of the bat captured on December 10, 1982, from the basement of Safdar-Jang Tomb, a historical monument in New Delhi. The fungus was not found in 581 other bats representing R. hardwickei hardwickei, three more insectivorous and one frugivorous species investigated from several sites in Delhi and New Delhi metropolitan areas. The identity of the isolate was based upon its macroscopic and microscopic cultural morphology, dimorphic character and verification of pathogenicity for white mice. It was further confirmed by determining the capacity of the isolate to produce the 'A' exoantigen specific for B. dermatitidis. The infected bat did not manifest any obvious clinical signs and symptoms of illness. Its visceral organs were free from macroscopic lesions, and histopathologically none of them including the liver, revealed any fungal elements or tissue response. B. dermatitidis was not found in any of the 34 samples of bat guano investigated by direct culture or mouse-inoculation technique. The results reinforce the available evidence for the endemic occurrence of B. dermatitidis in India and focus on the possible role of R. hardwickei hardwickei as a natural host or vector for this pathogen.     

全球皮炎外瓶霉感染流行现状的回顾性分析

目的分析皮炎外瓶霉感染的流行病学和临床特征,为提高皮炎外瓶霉感染的诊治水平提供科学依据。方法采用文献回顾和荟萃分析的方法,分析全球范围内已报道的皮炎外瓶霉感染病例的国籍、性别、年龄分布、危险因素、发病部位、临床表现、诊疗方法及预后等流行病学和临床特征。结果皮炎外瓶霉感染在免疫功能正常和免疫功能缺陷患者中均可发生,患者的男女性别比为1.10∶1.00,最常发病年龄段为51~60岁,肺(27.90%,17/61)为最常受累的器官,但不同地域的病例感染器官存在差异。约半数(47.54%,29/61)病例伴有各种免疫抑制的基础疾病或危险因素。皮炎外瓶霉感染的临床确诊主要依赖培养和分子鉴定,系统感染患者推荐联合伊曲康唑和特比萘芬作为抗真菌治疗方案。结论近年来皮炎外瓶霉感染的发病率在全球范围内呈上升趋势,肺部为系统感染患者最常受累的器官,诊断主要依赖于真菌培养。加强皮炎外瓶霉菌株的药敏监测和分子流行病学研究,对于提高皮炎外瓶霉感染的临床诊治水平非常必要。     

Effects of culture filtrates of Blastomyces dermatitidis on neutrophil locomotion

A potent chemotactic activity for neutrophils is detectable in liquid culture filtrates of Blastomyces dermatitidis. The production of this activity is medium-dependent and culture age-dependent. The highest levels of cytotaxin were produced in filtrates of B. dermatitidis grown in tissue culture medium 199 for three or more weeks. This factor(s) stimulates directed as well as random migration. It functions directly and independently of serum. It is stable at -20 degrees C and 56 degrees C, and has a molecular weight greater than 10000 daltons. These properties define a new microbial chemotactic factor.     

Use of ribosomal introns as new markers of genetic diversity in Exophiala dermatitidis

Exophiala dermatitidis is one of the prevalent black yeasts found as opportunistic pathogens or colonizers in humans. In the tropics its natural habitat is thought to be fruit surfaces and it is also found in the digestive system of fruit-eating animals. However, it has recently been abundantly isolated from human-made environments (steam baths, railway ties, dishwashers) in tropical and temperate climates. Two genotypes have been distinguished within this species: genotype A, mostly corresponding to strains isolated from patients, and genotype B, to strains isolated from the natural environment. In human-made environments, both genotypes A and B occur. A previous study suggested that one genotype had been selected for in the human host. In our study, the distribution of ribosomal insertions agrees with an ecological specialization of E. dermatitidis genotypes by showing a significantly higher frequency of ribosomal insertions in clinical strains in comparison to environmental ones. The characterization of these insertions shows that they correspond to introns of group IC or IE, the most frequent types within the fungal kingdom. These ribosomal group I introns could be used as new markers for populations of E. dermatitidis.     

Blastomycosis: a new endemic focus in Canada.

A survey of the 38 patients resident in Ontario from whose sputum, body fluids or tissues Blastomyces dermatitidis was cultured by our laboratory between 1970 and 1981 revealed a new endemic focus to the north and east of Lake Superior, where 20 of 27 traceable patients lived. Direct microscopy revealed B. dermatitidis in 90% of the cases. A lack of clinical awareness, however, had often resulted in a delay (average 15 weeks) in diagnosis. In two cases the disease was identified only at autopsy. About 80% of the patients survived.     

Native valve Aspergillus fumigatus endocarditis with blood culture positive and negative for galactomannan antigen. Case report and literature review

Native valve endocarditis caused by Aspergillus spp. is an uncommon disease with a high mortality rate. Generally, Aspergillus is isolated from affected valve in post-mortem or biopsy specimens. However, its isolation from blood cultures is exceedingly rare. We report a case of fungal endocarditis in a native mitral valve with the isolation of Aspergillus fumigatus both in valve vegetation and in blood culture bottles. The patient underwent valve replacement and antifungal treatment with voriconazole and caspofungin, but he died on post-operative day 45 with disseminated aspergillosis confirmed by necropsy. Paradoxically, galactomannan antigen detection in serum was negative. This is the third case of Aspergillus endocarditis with positive blood culture reported in the literature.     

Evaluation of the in vitro and in vivo dimorphism of Sporothrix schenckii, Blastomyces dermatitidis, and Paracoccidioides brasiliensis isolates after preservation in mineral oil

Morphological differentiation has commanded attention for its putative impact on the pathogenesis of invasive fungal infections. We evaluated in vitro and in vivo the dimorphism from mycelial to yeast-phase of Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis isolates, two strains for each species, preserved in mineral oil. S. schenckii strains showed typical micromorphology at 25 degrees C but one strain was unable to complete the dimorphic process in vitro. After in vivo passage through mice the strains had the ability to turn into yeast-like cells and to form colonies on brain-heart infusion medium at 36 degrees C. B. dermatitidis strains grew as dirty white to brownish membranous colonies at 25 degrees C and their micromorphology showed thin filaments with single hyaline conidia. At 36 degrees C the colonies did not differ from those grown at 25 degrees C, but produced a transitional micromorphology. P. brasiliensis strains grew as cream-colored cerebriform colonies at 25 degrees C showing a transitional morphology. B. dermatitidis and P. brasiliensis strains did not turn into yeast-like cells in vivo. The present results demonstrate that B. dermatitidis and P. brasiliensis strains were unable to complete the dimorphic process even after in vivo passage, in contrast to the S. schenckii strain.     

Evaluation of a selective enrichment technique for the isolation of Campylobacter pylori

To cultivate Campylobacter pylori from contaminated biopsy specimens, Brucella broth was supplemented with 10% fetal calf serum, 1% Vitox, 1000 units/ml polymyxin B sulfate, 10 micrograms/ml vancomycin, and 2 micrograms/ml amphotericin B. Pseudomonas aeruginosa, Candida albicans, and Enterococcus fecalis were cocultivated with C. pylori. All four strains of C. pylori were recoverable at 24 h. When 21 C. pylori strains were studied in pure culture, 86% grew in the selective enrichment medium. In a clinical study, the selective enrichment technique resulted in isolation of C. pylori from 50% of patient samples, compared with isolation from only 36% of samples with agar cultivation. The selective enrichment technique may be more sensitive than techniques currently employed to isolate C. pylori from gastric tissue.     

Population genetic structure of clinical and environmental isolates of Blastomyces dermatitidis, based on 27 polymorphic microsatellite markers

Blastomyces dermatitidis, a thermally dimorphic fungus, is the etiologic agent of North American blastomycosis. Clinical presentation is varied, ranging from silent infections to fulminant respiratory disease and dissemination to skin and other sites. Exploration of the population genetic structure of B. dermatitidis would improve our knowledge regarding variation in virulence phenotypes, geographic distribution, and difference in host specificity. The objective of this study was to develop and test a panel of microsatellite markers to delineate the population genetic structure within a group of clinical and environmental isolates of B. dermatitidis. We developed 27 microsatellite markers and genotyped B. dermatitidis isolates from various hosts and environmental sources (n=112). Assembly of a neighbor-joining tree of allele-sharing distance revealed two genetically distinct groups, separated by a deep node. Bayesian admixture analysis showed that two populations were statistically supported. Principal coordinate analysis also reinforced support for two genetic groups, with the primary axis explaining 61.41% of the genetic variability. Group 1 isolates average 1.8 alleles/locus, whereas group 2 isolates are highly polymorphic, averaging 8.2 alleles/locus. In this data set, alleles at three loci are unshared between the two groups and appear diagnostic. The mating type of individual isolates was determined by PCR. Both mating type-specific genes, the HMG and α-box domains, were represented in each of the genetic groups, with slightly more isolates having the HMG allele. One interpretation of this study is that the species currently designated B. dermatitidis includes a cryptic subspecies or perhaps a separate species.     

Effect of a local immune reaction on peripheral blood polymorphonuclear neutrophil microbicidal function: studies with fungal targets

Peripheral blood polymorphonuclear neutrophils (PMN) from mice immunized with Blastomyces dermatitidis and then stimulated locally (intraperitoneally, ip) with B. dermatitidis antigen had enhanced killing of B. dermatitidis in vitro (54.4 +/- 19.49 of inoculum) compared to nonimmune mice (32.7 +/- 8.7%; P less than 0.02), nonimmune mice given antigen ip (30.6 +/- 14.0%; P less than 0.05), or immune mice not given antigen ip (15.4 +/- 9.9%; P less than 0.01). Peripheral blood PMN from all four groups had marked killing ability against Candida albicans (91.8-99.3% of inoculum). That the killing of B. dermatitidis was due to PMNs was demonstrated by lack of killing by isolated peripheral blood mononuclear cells from all four groups. A local immune reaction can result in enhancement of PMN fungicidal activity, and this is reflected even in peripheral blood PMN. We hypothesize this is an important component of normal host defenses against fungal infection, and likely other microbial infections. Enhancement of PMN microbicidal function by the soluble mediators presumed to be responsible for the effects observed may be an approach to immunomodulating therapy or prophylaxis of infection.     

An integrated approach for increasing the survival of autologous fat grafts in the treatment of contour defects

Autologous fat grafting as a technique to correct soft-tissue defects is a controversial subject. The high percentage of fat resorption and the resulting need for additional grafting considerably reduce the value of this method. The purpose of this study was to evaluate the clinical application of tissue-culturing methodology in the handling of the lipocyte aspirate in an endeavor to improve the survival rate and therefore the take of the grafted lipocytes. The method consists of syringe aspiration of the lipocytes from the donor site, isolation of intact lipocytes by gentle centrifugation, suspension of the aspirate in an enriched cell culture medium, and injection of the cell suspension into preformed subdermal tunnels. A number of media were tested and shown to prolong the survival of lipocytes ex vivo using fluorescent acridine orange stain. Implementing the integrated cell culture techniques increased the lipocytes' viability, as indicated in clinical evaluation in which the amount of graft take ranged between 50 and 90 percent. The results of 15 patients with varied types of cases who were operated on using this new methodology show that the tissue defect was filled and remained so in postoperative follow-ups of 6 to 24 months. A three-dimensional CAT scan-aided evaluation method was developed and used in one of four case histories presented herein.     

Cutaneous blastomycosis. An imported case with good response to itraconazole

BackgroundBlastomycosis is a subacute or chronic deep mycosis caused by a dimorphic fungus called Blastomyces dermatitidis, which generally produces a pulmonary form of the disease and, to a lesser extent, extra-pulmonary forms such as cutaneous, osteoarticular and genitourinary, among others. Cutaneous blastomycosis is the second clinical presentation in frequency. It is considered as primary when it begins by inoculation of the fungus due to traumas, and secondary when the lung fails to contain the infection.Case-reportWe present the case of a 57 year-old male who had a 5 year-history of an irregularly shaped verrucous infiltrative plaque related to and insect bite and posterior trauma due to the manipulation of the lesion. B. dermatitidis was identified using direct examination, stains, isolation in culture media, histopathology, and molecular studies. An antifungal susceptibility test was performed using method M38-A2 (CLSI). Clinical and mycological cure was achieved with itraconazole.ConclusionsThis cutaneous blastomycosis case acquired in the United States (Indianapolis) is rather interesting and looks quite similar to other mycoses such as coccidioidomycosis or sporotrichosis. The presented case shows one of the multiple issues concerning migration between neighboring countries.     

An Imported Case of Blastomyces Dermatitidis Infection in Mexico

Blastomycosis is an acute or chronic primary infection of the respiratory system, endemic in North America (United States of America and Canada), Africa and Asia. We report a case in Mexico, in a three years old child who had been born in California and lived in Chicago, U.S.A. The patient presented pulmonary symptoms prior to development of a skin ulcer. Blastomyces dermatitidis was identified by mycological and molecular procedures. The patient was successfully treated with amphotericin B, oral ketoconazole and itraconazole.     

Monosaccharide and Chitin Content of Cell Walls of Histoplasma capsulatum and Blastomyces dermatitidis

Cell walls of Histoplasma capsulatum and Blastomyces dermatitidis, obtained by mechanical breakage of yeast- and mycelial-phase cultures, were lipid-extracted and then fractionated with ethylenediamine. Unextracted cell walls, lipid-extracted cell walls, and the three fractions resulting from ethylenediamine treatment were examined for monosaccharide and chitin content. The yeast-phase cell walls of five strains of H. capsulatum fell into two categories, designated chemotypes I and II, one of which, chemotype II, was similar to yeast-phase cell walls derived from three strains of B. dermatitidis. H. capsulatum chemotype I cell walls were characterized by lower content of material soluble in ethylenediamine, higher chitin content, and lower monosaccharide content than H. capsulatum chemotype II or B. dermatitidis cell walls. Approximately 80% of the monosaccharides of chemotype I cell walls was combined in forms susceptible to attack by mild acid hydrolysis, compared with about 50% of the monosaccharides of chemotype II and B. dermatitidis. H. capsulatum and B. dermatitidis yeast-phase cell walls could be distinguished, however, by their susceptibility to attack by a crude enzyme system derived from a Streptomyces sp. incubated with chitin as the only carbon source. Both glucose and acetylglucosamine were released from H. capsulatum cell walls, regardless of chemotype, during enzymatic hydrolysis, whereas only acetylglucosamine was released from B. dermatitidis yeast-phase cell walls. Mycelial-phase cell walls of H. capsulatum and B. dermatitidis were characterized by lower content of material soluble in ethylenediamine, higher proportions of mannose, and lower chitin content than their respective yeast phases. Glucose and acetylglucosamine were both released from all mycelial-phase cell walls, whether H. capsulatum or B. dermatitidis, by the crude enzyme system.     

Conditions to improve expansion of human mesenchymal stem cells based on rat samples

AIM: To improve the isolation and expansion of human marrow-derived mesenchymal stem cells (MSCs) based on rat samples. METHODS: Based on the fact that rat MSCs are relatively easy to obtain from a small aspirate, bone marrow-derived MSCs from rat were cultured and characterized to set up the different protocols used in this study. Then, accordingly, almost the same protocols were performed on human healthy bone marrow samples, after obtaining approval of the ethics committee and gaining informed consent. We used different protocols and culture conditions, including the type of basal media and the culture composition. The MSCs were characterized by immunophenotyping and differentiation. RESULTS: There was no difference in morphology and proliferation capacity between different culture media at the first passage. During the 5-7th passages, the cells gradually lost their morphology and proliferation potential on Dulbecco’s modified Eagle’s medium (DMEM) high glucose and α modified Eagle’s medium. Although the cells expanded rapidly for up to 10 passages on DMEM low glucose containing 10% to 15% fetal calf serum (FCS), their proliferation was arrested without change in morphology and differentiation capacity at the third passage on 5% FCS. Flow cytometric analysis and functional tests confirmed that more than 90% of marrow cells which were isolated and expanded by our selective protocols were MSCs. CONCLUSION: We improved the isolation and expansion of human bone marrow derived MSCs, based on rat sample experiments, for further experimental and clinical use.     

裸鼠巴斯德氏菌病的诊断

目的对某实验动物中心裸鼠发病死亡的原因进行诊断。方法对发病裸鼠进行病理组织学和细菌学检查以及血清学试验。结果根据临床症状、病理剖检、细菌分离培养、生化鉴定和血清凝集试验,确诊为嗜肺巴斯德氏菌感染导致的细菌性疾病。药敏试验结果表明,分离菌株对阿莫西林／克拉维酸、阿奇霉素、复方新诺明敏感,而对红霉素已经产生耐药性。结论为巴斯德氏菌病原鉴定与疾病诊断提供了科学依据。     

Adiaspiromycosis of human skin caused by Emmonsia crescens

Two cases of cutaneous adiaspiromycosis by Emmonsia crescens are reported. This is the first human skin infection by this species and is the first report of its kind in man from India. In the first patient, the agent was demonstrated in KOH mounts, histology and culture from irregular, pigmented skin plaques on the right gluteal area. The lesion also contained calcium. In the second patient the fungus was demonstrated histologically in a knee lesion. The agent had elicited a histiocytic and giant cell reaction in the dermis in both cases. The first patient suffered from anaemia and epilepsy and the second suffered from nephropathy with chyluria. The skin lesions were surgically excised with skin grafting in the first patient.     

Heterogeneity of bronchial endothelial cell permeability

In vivo models of airway inflammation suggest that most protein transudation occurs from bronchial microcirculation. However, due to technical limitations in the isolation and culture of bronchial endothelial cells, most studies of lung vascular permeability have focused on pulmonary endothelium. Thus conditions for culture of sheep bronchial artery endothelial cells (BAEC) and bronchial microvascular endothelial cells (BMVEC) were established. The bronchial artery and the mainstem bronchi, stripped of epithelium, were dissected, and endothelial cells were isolated by enzymatic treatment. BAEC and BMVEC demonstrated positive staining for factor VIII-related antigen, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled low-density lipoprotein, and PECAM-1. Radioligand binding studies confirmed equivalent numbers of bradykinin B(2) receptors on BAEC and BMVEC. Permeability of BAEC and BMVEC was determined after treatment with bradykinin and thrombin by comparing the translocation of FITC-dextran (mol wt 9,500) across confluent monolayers (n = 10-12). Bradykinin caused a maximal increase in permeability in BAEC (165% increase) and BMVEC (144% increase) by 15 min compared with vehicle controls. Thrombin treatment altered BMVEC permeability only, reaching a maximal response at 60 min (109% increase). These results demonstrate bronchial endothelial cell heterogeneity and establish methods to determine intracellular mechanisms contributing to airway disease in relevant cell systems.