非侵袭性真菌性鼻窦炎的实验室诊断及致病菌分析

目的探讨非侵袭性真菌性鼻窦炎的实验室诊断方法,分析其致病菌,为鼻窦炎合并真菌感染的临床诊断、治疗提供依据。方法对我院临床及鼻内镜下所诊断的10例真菌性鼻窦炎患者,鼻内镜手术时直接吸取病变的鼻窦黏膜及窦腔内容物,通过直接镜检、真菌培养、传统鉴定及分子生物学鉴定和组织病理学检查对其进行检查。结果 10例病例中,直接镜检阳性者8例;病理学检查可见真菌菌丝或者孢子者8例;接种培养及基因鉴定阳性者5例(感染菌株包括2例烟曲霉复合体、1例杂色曲霉、1例枝孢样枝孢霉、1例帚霉)。不同方法检测出的阳性病例并非完全重叠。结论真菌镜检、真菌培养、真菌分子生物学鉴定、组织病理学检查在诊断真菌感染时可以互补,有助于明确诊断及发现新菌株。     

Aspergillosis in German cockroach Blattella germanica (L.) (Blattoidea: Blattellidae)

Natural infection of Aspergillus flavus was observed in adults of Blattella germanica. Though the adult insects exhibited no external symptoms, they became hypoactive and later died. The dead and experimentally infected insects repeatedly yielded Aspergillus flavus in culture on Czapek's medium. Direct microscopic observation of the tissues of infected insects revealed fungal material. The blood films stained with Giemsa stain showed granulocytes (GRs) engulfing fungal hyphae. A remarkable increase in GR and plasmatocyte (PL) counts occurred in differential haemocyte counts (DHCs) of the infected insects. Two main types of immunological responses of the insect noticed were phagocytosis and encapsulation. DHC showed maximum involvement of GRs and PLs in immune mechanism. This revised version was published online in June 2006 with corrections to the Cover Date.     

The cotton rat (Sigmodon hispidus) is a permissive small animal model of human metapneumovirus infection, pathogenesis, and protective immunity

Human metapneumovirus (hMPV) is a newly described paramyxovirus that is an important cause of acute respiratory tract disease. We undertook to develop a small animal model of hMPV infection, pathogenesis, and protection. Hamsters, guinea pigs, cotton rats, and nine inbred strains of mice were inoculated intranasally with hMPV. The animals were sacrificed, and nasal and lung tissue virus yields were determined by plaque titration. None of the animals exhibited respiratory symptoms. The quantity of virus present in the nasal tissue ranged from 4.6 x 10(2) PFU/gram tissue (C3H mice) to greater than 10(5) PFU/gram (hamster). The amount of virus in the lungs was considerably less than in nasal tissue in each species tested, ranging from undetectable (<5 PFU/g; guinea pigs) to 1.8 x 10(5) PFU/gram (cotton rat). The peak virus titer in cotton rat lungs occurred on day 4 postinfection. hMPV-infected cotton rat lungs examined on day 4 postinfection exhibited histopathological changes consisting of peribronchial inflammatory infiltrates. Immunohistochemical staining detected virus only at the luminal surfaces of respiratory epithelial cells throughout the respiratory tract. hMPV-infected cotton rats mounted virus-neutralizing antibody responses and were partially protected against virus shedding and lung pathology on subsequent rechallenge with hMPV. Viral antigen was undetectable in the lungs on challenge of previously infected animals. This study demonstrates that the cotton rat is a permissive small animal model of hMPV infection that exhibits lung histopathology associated with infection and that primary infection protected animals against subsequent infection. This model will allow further in vivo studies of hMPV pathogenesis and evaluation of vaccine candidates.     

Experimental Sendai virus infection in laboratory rats. II. Pathology and immunohistochemistry

Intranasal inoculation of 5 to 8 week old specific pathogen-free Sprague-Dawley rats with 5 X 10(3) egg infectious doses of Sendai virus resulted in severe rhinitis, bronchiolitis and alveolitis. The most severe rhinitis occurred on postinoculation (PI) days 4-6, and pneumonia on day 4. Rhinitis and pneumonia persisted to PI day 21, with peribronchial lymphoid infiltration detectable at PI day 42. Immunohistochemical studies showed that Sendai virus antigens were present primarily in columnar epithelial cells of the respiratory mucosa of the nasal cavity and in bronchiolar and alveolar epithelium. Antigen was first detectable at PI day 1, was most prominent at days 3-4 and was undetectable after day 7. More antigen could be seen in the nasal mucosa than in the lung at any stage in the infection. These studies show that Sendai virus by itself is capable of evoking severe, although transient, rhinitis and pneumonia in laboratory rats free of other significant pathogens.     

A Case of Cunninghamella bertholettiae Rhino-cerebral Infection in a Leukaemic Patient and Review of Recent Published Studies

Cunninghamella bertholletiae infection occurs most frequently in neutropenic patients affected by haematological malignancies, is associated with an unfavourable outcome. We report a case of rhino-mastoidal fungal infection in a leukaemic patient. Bioptical tissue cultures yield the isolation of a mould with typical properties of Cunninghamella species. Liposomal amphotericin B (L-Amb) therapy combined with surgical intervention brought the lesion to recovery. Nevertheless, the patient died 14 days after bone marrow transplantation (BMT) from bacterial sepsis. Mastoiditis was documented at CT-scan. The conditioning regimen probably caused the reactivation of the Cunninghamella infection that led to the patient's fatal outcome; fungal hyphae were detected after autopsy of brain and lung tissue.     

Human polymorphonuclear leukocytes inhibit Aspergillus fumigatus conidial growth by lactoferrin-mediated iron depletion

Aspergillus fumigatus, a common mold, rarely infects humans, except during prolonged neutropenia or in cases of chronic granulomatous disease (CGD), a primary immunodeficiency caused by mutations in the NADPH oxidase that normally produces fungicidal reactive oxygen species. Filamentous hyphae of Aspergillus are killed by normal, but not CGD polymorphonuclear leukocytes (PMN); however, the few studies on PMN-mediated host defenses against infectious conidia (spores) of this organism have yielded conflicting results, some showing that PMN do not inhibit conidial growth, with others showing that they do, most likely using reactive oxygen species. Given that CGD patients are exposed daily to hundreds of viable A. fumigatus conidia, yet considerable numbers of them survive years without infection, we reasoned that PMN use ROS-independent mechanisms to combat Aspergillus. We show that human PMN from both normal controls and CGD patients are equipotent at arresting the growth of Aspergillus conidia in vitro, indicating the presence of a reactive oxygen species-independent factor(s). Cell-free supernatants of degranulated normal and CGD neutrophils both suppressed fungal growth and were found to be rich in lactoferrin, an abundant PMN secondary granule protein. Purified iron-poor lactoferrin at concentrations occurring in PMN supernatants (and reported in human mucosal secretions in vivo) decreased fungal growth, whereas saturation of lactoferrin or PMN supernatants with iron, or testing in the presence of excess iron in the form of ferritin, completely abolished activity against conidia. These results demonstrate that PMN lactoferrin sequestration of iron is important for host defense against Aspergillus.     

Selective staining of fungal hyphae in parasitic and symbiotic plant-fungus associations

A cytochemical method for light microscopical studies is described which allows the specific detection of fungal hyphae in plant-fungus associations: e.g. lichens, mycorrhiza, or fungal infections of plant tissue. The specimens were fixed and embedded in epoxy resin by a standard protocol for electron microscopy. Semithin sections were successively incubated with fluorescein isothiocyanate labelled wheat germ agglutinin (FITC-WGA) and calcofluor white (CW). FITC-WGA stained exclusively the fungal cell walls while CW stained both the fungal and the plant cell walls. Therefore, FITC-WGA is an excellent marker for the fungal hyphae.     

Rhinitis,Ocular, Throat and Dermal Symptoms,Headache and Tiredness among Students in Schools from Johor Bahru,Malaysia: Associations with Fungal DNA and Mycotoxins in Classroom Dust

There are few studies on rhinitis and sick building syndrome (SBS) among students in tropical countries. We studied associations between levels of five fungal DNA sequences, two mycotoxins (sterigmatocystin and verrucarol) and cat allergen (Fel d 1) levels in schools and rhinitis and other weekly SBS symptoms in the students. Fungal DNA was measured by quantitative PCR and cat allergen by ELISA. Pupils (N = 462) from eight randomly selected schools in Johor Bahru, Malaysia participated (96%). Dust samples were collected by cotton swabs and Petri dishes exposed for one week. None of the schools had a mechanical ventilation system, but all classrooms had openable windows that were kept open during lectures and indoor CO₂ levels were low (mean 492 ppm; range 380–690 ppm). Weekly nasal symptoms (rhinitis) (18.8%), ocular (11.6%), throat (11.1%), dermal symptoms, headache (20.6%) and tiredness (22.1%) were common. Total fungal DNA in swab samples was associated with rhinitis (p = 0.02), ocular symptoms (p = 0.009) and tiredness (p = 0.001). There were positive associations between Aspergillus versicolor DNA in Petri dish samples, ocular symptoms (p = 0.02) and tiredness (p = 0.001). The level of the mycotoxin verrucarol (produced by Stachybotrys chartarum) in swab samples was positively associated with tiredness (p = 0.04). Streptomyces DNA in swab samples (p = 0.03) and Petri dish samples (p = 0.03) were negatively associated with tiredness. In conclusion, total fungal contamination, measured as total fungal DNA) in the classrooms, Aspergillus versicolor and verrucarol can be risk factors for rhinitis and SBS symptoms among students in the tropical country Malaysia.     

Fungal endophytes in Astromyelon-type (Sphenophyta,Equisetales, Calamitaceae) roots from the Upper Pennsylvanian of France

A distinctive fungal endophyte, Cashhickia acuminata nov. gen. et sp., is described from permineralized calamite roots from the Upper Pennsylvanian Grand-Croix cherts of France. Heavily infected roots contain numerous intracellular hyphae in the outer cortex that arise from a meshwork-like mycelium extending between cortical cells. All intracellular hyphae are oriented toward the root center; none occur on the inner periclinal host cell walls. Other roots of the same type show localized infection by this fungus in which isolated cortical cells contain or give rise to intracellular fungal growth. Within the cortical cells are host responses in the form of callosities that indicate the roots were alive at the time of infection. Other endophytes are present in the same host tissue but are less frequent. The discovery of this association provides the first detailed account on the morphology of a Carboniferous fungal root endophyte, as well as the spatial distribution within the host, and infection pathways within the cortical tissues.     

Phoma (Peyronellaea) as zoopathogen.

Two case reports add to the rare recorded instances of infection of man and other animals by Phoma or Phoma-like molds. Phoma cava was consistently cultured from aural dermatitis of a white-tailed deer (Odocoileus virginianus), and tissue sections of the ear revealed fragments of apparent pycnidial walls, as well as many septate hyphae. In the second instance, numerous Phoma-like pycnidia occurred in and upon the hairs of a young child, in a lesion initiated by Trichophyton infection.     

Intratracheal exposure of rats to Aspergillus fumigatus spores isolated from sawmills in Sweden.

Five strains of Aspergillus fumigatus (A, B, D, H, and K) isolated from sawmills were used to expose groups of three rats by intratracheal intubation. The dose was 10(9) spores per rat. At 48 h after administration, two rats from the D group and all rats from the K group died with symptoms of strong dyspnea and tachypnea. At 72 h postadministration and after, some animals showed mild to moderate dyspnea and tachypnea. Autopsies of all animals were performed, including a histopathological examination of the lungs. At 72 h after administration, two distinct morphological groups were identified histopathologically. Severe necrotizing pneumonia characterized by the presence of abundant fungal hyphae was seen in animals that died spontaneously within 48 h postadministration and rats with bronchopneumonia and was characterized by the presence of numerous fungal spores. There was an obvious difference in pathogenicity among the strains of A. fumigatus. Strains D and K were more pathogenic, and only the rats exposed to these strains showed the presence of fungal hyphae in the lungs. The mycotoxin gliotoxin that is produced by A. fumigatus and has antiphagocytic activity was not detected in the spores from any of the A. fumigatus strains.     

Intratracheal exposure of rats to Aspergillus fumigatus spores isolated from sawmills in Sweden

Five strains of Aspergillus fumigatus (A, B, D, H, and K) isolated from sawmills were used to expose groups of three rats by intratracheal intubation. The dose was 10(9) spores per rat. At 48 h after administration, two rats from the D group and all rats from the K group died with symptoms of strong dyspnea and tachypnea. At 72 h postadministration and after, some animals showed mild to moderate dyspnea and tachypnea. Autopsies of all animals were performed, including a histopathological examination of the lungs. At 72 h after administration, two distinct morphological groups were identified histopathologically. Severe necrotizing pneumonia characterized by the presence of abundant fungal hyphae was seen in animals that died spontaneously within 48 h postadministration and rats with bronchopneumonia and was characterized by the presence of numerous fungal spores. There was an obvious difference in pathogenicity among the strains of A. fumigatus. Strains D and K were more pathogenic, and only the rats exposed to these strains showed the presence of fungal hyphae in the lungs. The mycotoxin gliotoxin that is produced by A. fumigatus and has antiphagocytic activity was not detected in the spores from any of the A. fumigatus strains.     

Fungal invasion of normally non-phagocytic host cells

Many fungi that cause invasive disease invade host epithelial cells during mucosal and respiratory infection, and subsequently invade endothelial cells during hematogenous infection. Most fungi invade these normally non-phagocytic host cells by inducing their own uptake. Candida albicans hyphae interact with endothelial cells in vitro by binding to N-cadherin on the endothelial cell surface. This binding induces rearrangement of endothelial cell microfilaments, which results in the endocytosis of the organism. The capsule of Cryptococcus neoformans is composed of glucuronoxylomannan, which binds specifically to brain endothelial cells, and appears to mediate both adherence and induction of endocytosis. The mechanisms by which other fungal pathogens induce their own uptake are largely unknown. Some angioinvasive fungi, such as Aspergillus species and the Zygomycetes, invade endothelial cells from the abluminal surface during the initiation of invasive disease, and subsequently invade the luminal surface of endothelial cells during hematogenous dissemination. Invasion of normally non-phagocytic host cells has different consequences, depending on the type of invading fungus. Aspergillus fumigatus blocks apoptosis of pulmonary epithelial cells, whereas Paracoccidioides brasiliensis induces apoptosis of epithelial cells. This review summarizes the mechanisms by which diverse fungal pathogens invade normally non-phagocytic host cells and discusses gaps in our knowledge that provide opportunities for future research.     

Colonization and Degradation of Thermally Oxidized High-Density Polyethylene by Aspergillus niger (ITCC No. 6052) Isolated from Plastic Waste Dumpsite

Plastic materials, particularly polyethylene, are the potential source of environmental pollution. In the present study, a fungal strain was isolated from plastic waste dumpsites capable of adhering to high-density polyethylene (HDPE) surface. The fungal strain was identified as Aspergillus niger (ITCC no. 6052). A visible increase in the growth of the fungi was observed on the surface of the polyethylene when cultured in minimal medium at 30°C and 120 rpm, for 1 month. Approximately 3.44% reduction (gravimetrically) in mass and 61% reduction in tensile strength of polyethylene was observed after 1 month of incubation with fungal isolate. Scanning electron microscope analysis showed hyphael penetration and cracks on the surface of polyethylene. A thick network of fungal hyphae forming a biofilm was also observed on the surface of the plastic pieces. The efficient biofilm formation on polyethylene surface by Aspergillus niger (ITCC no. 6052) is attributed to its high cell surface hydrophobicity. This study indicated that Aspergillus niger (ITCC no. 6052) has ability to degrade thermally oxidized polyethylene.     

Chronic sialodacryoadenitis virus (SDAV) infection in athymic rats.

Sialodacryoadenitis virus (SDAV) was detected in athymic rats subcutaneously implanted with human tumor cell lines. Clinical signs included sneezing, dyspnea, weight loss and death. Necropsy revealed both upper and lower respiratory tract disease from which Staphylococcus aureus, Pasteurella pneumotropica and Pseudomonas aeruginosa were recovered. Histopathological changes consisted of suppurative rhinitis and bronchopneumonia. Lesions characteristic of SDAV infection were found in lacrimal and salivary glands, and viral antigens were detected in the salivary glands and respiratory tract by immunohistochemistry. Submaxillary salivary gland. Harderian gland and lung homogenates from affected athymic rats were inoculated intranasally into euthymic rats as a rat antibody production test. All euthymic rats seroconverted to SDAV. Seroconversion to SDAV was demonstrated in consecutive pairs of sentinel euthymic rats housed for 6 months with infected athymic rats. Inoculation of supernatants of the original tumor cell lines into euthymic rats did not result in seroconversion. The source of the virus was not determined. In this study, spontaneously acquired SDAV infection persisted for at least 6 months in athymic rats.     

Spread and change in stress resistance of Shiga toxin-producing Escherichia coli?O157 on fungal colonies

To elucidate the effect of fungal hyphae on the behaviour of Shiga toxin-producing Escherichia coli (STEC) O157, the spread and change in stress resistance of the bacterium were evaluated after coculture with 11 species of food-related fungi including fermentation starters. Spread distances of STEC O157 varied depending on the co-cultured fungal species, and the motile bacterial strain spread for longer distances than the non-motile strain. The population of STEC O157 increased when co-cultured on colonies of nine fungal species but decreased on colonies of Emericella nidulans and Aspergillus ochraceus. Confocal scanning microscopy visualization of green fluorescent protein-tagged STEC O157 on fungal hyphae revealed that the bacterium colonized in the water film that existed on and between hyphae. To investigate the physiological changes in STEC O157 caused by co-culturing with fungi, the bacterium was harvested after 7 days of co-culturing and tested for acid resistance. After co-culture with eight fungal species, STEC O157 showed greater acid resistance compared to those cultured without fungi. Our results indicate that fungal hyphae can spread the contamination of STEC O157 and can also enhance the stress resistance of the bacteria.     

鼻内镜手术合并大环内酯类抗生素治疗鼻窦炎43例

目的：评价鼻内镜手术治疗合并术后利用大环内酯类抗生素治疗鼻窦炎的疗效,探讨鼻窦炎患者治疗前后血清中IL-17及其受体的表达及血清中总IgE的变化。方法：鼻窦内窥镜行病侧上颌寞自然口扩大及下鼻道开窗双进路,彻底清除鼻腔、鼻窦病变组织,术后采用大环内酯类抗生素治疗;采用酶联免疫法检测30例患者治疗前后血清中IL-17,IL-17R及IgE的含量。结果：治疗30例,其中术后病理证实鼻寞真菌球20例,治愈20例;曲霉菌感染23例,治愈10例,6个月随访无复发;与只治疗前相比,手术治疗后鼻窦炎患者血清IL-17,IL-17R及IgE的含量均明显降低。结论：鼻内镜手术是治疗真菌性鼻一鼻窦炎的主要方法,IL-17和IL-17R均参与鼻窦炎的发病过程,可作为诊断鼻窦炎的新指标,鼻内镜术后使用大环内酯类抗生素进一步巩固治疗,可降低复发率。     

Biodegradation of ivory (natural apatite): possible involvement of fungal activity in biodeterioration of the Lewis Chessmen

Fungal biodeterioration of ivory was investigated with in vitro inoculation of samples obtained from boar and walrus tusks with the fungi Aspergillus niger and Serpula himantioides, species of known geoactive abilities. A combination of light and scanning electron microscopy together with associated analytical techniques was used to characterize fungal interactions with the ivory, including changes in ivory composition, dissolution and tunnelling, and the formation of new biominerals. The research was aimed at providing further understanding of the potential roles of fungi in the colonization and deterioration of ivory in terrestrial environments, but also contributes to our knowledge regarding the possible origins of the surface damage observed on early medieval sculptures made largely from walrus tusks, referred to as ‘the Lewis hoard of gaming pieces’, that were presumably produced for playing chess. The experiments have shown that the possibility of damage to ivory being caused by fungi is realistic. Scanning electron microscopy revealed penetration of fungal hyphae within cracks in the walrus tusk that showed also widespread tunnelling by fungal hyphae as well as ‘fungal footprints’ where the surface was etched as a consequence of mycelial colonization. Similar phenomena were observed with boar tusk ivory, while production of metabolites could lead to complete dissolution of the sample. Colonization of ivory and/or exposure to fungal activity lead to extensive secondary biomineral formation, and this was identified as calcium oxalate, mainly as the monohydrate, whewellite.     

Effect of a virus on accumulation of a tissue-specific cell-surface protein of the fungus Cryphonectria (Endothia) parasitica.

Hypovirulence and decreased sporulation of the plant pathogenic fungus Cryphonectria (Endothia) parasitica is caused by double-stranded (ds)RNAs. These symptoms of dsRNA infection are correlated with down-regulation of at least nine major fungal polypeptides. One of the regulated polypeptides was purified to homogeneity and antibody to it was prepared. This polypeptide (cryparin) has a -glycine-serine-repeating sequence near the amino-terminal end that is typical of structural proteins and has properties of a lectin. Antibody-staining showed that this 18.6-kDa polypeptide is specific to aerial hyphae and fruiting bodies and that it accumulates in large amounts on hyphal cell surfaces. The dsRNA affects accumulation of this protein, both in the fugal hyphae and in the growth medium. Cryparin is similar in physical properties to those of the putative phytotoxin cerato-ulmin produced by the Dutch elm disease fungus. Toxicity of cryparin is not detectable, but the striking similarities between the physical properties and locations of accumulation of cryparin and cerato-ulmin in fungal fruiting structures suggest either conservation of structure or convergent evolution in function of these two proteins.     

Serology of Aspergillus granuloma

A total of 21 cases of granuloma caused by Aspergillus species were encountered during the period 1972–79. The organs involved were nasal and paranasal sinuses, brain, orbit, subcutaneous tissue of cheek, lungs and endocardial valve in the decreasing order of their frequency. Aspergillus flavus was the main etiological agent. Immunodiffusion tests with various Aspergillus species as antigen showed a positivity of 17 out of 18 (94.4 per cent) cases. The role of precipitins in diagnosis and prognosis of the disease has been discussed.