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PubMed1993

Detection and differentiation of fungi in clinical specimens using polymerase chain reaction (PCR) amplification and restriction enzyme analysis.

We have developed a method for processing and detecting fungi in clinical specimens using the polymerase chain reaction (PCR) methodology. This PCR amplification of a segment of a ribosomal DNA gene results in a 310 bp product. The gene sequences used as amplimers have been highly conserved throughout the fungal kingdom and positive PCR results have been obtained for all genera and species of fungi tested (n = 42). Neither human nor a variety of pathogenic bacteria (n = 24) gave an amplified product. This PCR method can detect as few as 15 cells of Candida albicans in clinical specimens following a simple processing procedure. The sensitivity of the PCR for detecting other fungi remains to be determined. Analysis of the 310 bp amplified product, following digestion with the restriction enzyme HaeIII, can be used to further characterize the identity of the fungus involved into the following five groups: (i) Candida species and closely related yeasts; (ii) Cryptococci and Trichosporon species; (iii) Aspergilli and clinically related septate molds; (iv) the Zygomycetes; and (v) the dimorphic fungi.

Base Sequence↗
PubMed1998

Biodegradation of chlorpyrifos by either single or combined cultures of some soilborne plant pathogenic fungi.

Biodegradation of chlorpyrifos was studied in liquid culture media amended with either single or combined eight different plant pathogenic fungi isolated from the continuous cropping wheat fields. The average recovery of chlorpyrifos from the liquid media was found to be 86.1%. The detection limit of chlorpyrifos by the analytical method used was 19 ppb. Data showed that the growth of mixed fungi at concentrations up to 200 ppm of chlorpyrifos was higher than in the control treatment. Chlorpyrifos concentrations declined in the medium of combined fungi more than it did in the medium of any single fungus with increase in the incubation period. The amount of chlorpyrifos recovered was 79.8 ppm (39.9%) in the combined fungal cultures after 21 days. However, those recovered from the media of Fusarium graminearum, F. oxysporum, Rhizoctonia solani, Cladosporium cladosporiodes, Cephalosporium sp., Trichoderma viridi, Alternaria alternata, and Cladorrhinum brunnescens, ranged from 48.0 to 74.8%. The half-life value (T1/2) for chlorpyrifos was 15.8 day in the medium amended with mixed fungi. However, for the single cultures it ranged from 19.3 to 33.0 day.

Analysis of Variance↗
PubMed2003

Inactivation of fungi associated with barley grain by gaseous ozone.

The use of gaseous ozone as a fungicide to preserve stored barley was studied. The effects of the following operating parameters on the fungicidal efficacy of ozone were examined: 1) the applied ozone dose, 2) ozonation time, 3) water activity of barley, and 4) temperature of barley. The effect of ozonation on germination of barley was also investigated. The experimental results showed that ozone was very effective in inactivation of fungi associated with the barley regardless of whether the fungi were in the forms of spores or mycelia. However, the mycelia were less resistant to ozone. With 5 minutes of ozonation, 96% of inactivation were achieved for spores as well as for mixtures of spores and small amount of mycelia by applying 0.16 and 0.10 mg of ozone/(g barley) x min, respectively. In addition, for sealed storage silos, inactivation of fungi continued when the ozone-containing gas was held inside the silos following a continuous ozone supply. The experimental results also revealed that increases in water activity and temperature of barley enhanced the fungicidal efficacy of ozone. Results of this study also indicated that the inactivation processes could be controlled by simply monitoring the exit ozone from the reactor instead of performing the time-consuming microbial examination. This finding would make the application of ozone in the preservation of cereal grains easier, simpler, and more practically applicable. The experimental results demonstrated that although ozonation above certain strength may reduce barley germination, inactivation of fungi was achieved with ozonation strengths far below the critical point.

Dose-Response Relationship, Drug↗
PubMed1996

The relationship of endophytic fungi to the gametophyte of the fern Schizaea pusilla.

Schizaea pusilla is a rare and threatened fern restricted in North America to acidic bogs of Nova Scotia, Newfoundland, and New Jersey. The gametophyte lives in close association with two endophytic fungi. To characterize the nature of this fern's relationship with these fungi, we introduced axenic gametophytes to bog soil for colonization. Following colonization, the endophytic fungi were isolated and reintroduced to axenic gametophytes. The gametophytes introduced to bog soil were colonized by an aseptate fungus that formed vesicles and arbuscules within the gametophyte. However, culture of colonized gametophytes produced two fungal isolates: an aseptate fungus (fungus B) and a septate fungus (fungus A). Upon reintroduction of fungal isolates to axenically grown gametophytes, the aseptate fungus demonstrated a positive growth response to the presence of the gametophytes and colonized the gametophytes without harm to the host. The septate fungus did not exhibit any specific recognition but contacted the gametophytes randomly, leaving a large percentage of the host nonviable. We propose that the relationship of the septate fungus to the gametophyte of S. pusilla is nonmycorrhizal while the relationship of the aseptate fungus to the gametophyte is mycorrhizal. Furthermore, based on lack of nutrient availability in local soils, formation of specialized structures in the gametophyte for harboring fungi, and dependence of the fern on fungal presence for completion of its life cycle, we propose that S. pusilla maintains an obligatory relationship with the aseptate mycorrhizal fungus.

Calcium↗
PubMed2002

The ability of hospital ventilation systems to filter Aspergillus and other fungi following a building implosion.

OBJECTIVES: To assess the ability of hospital air handling systems to filter Aspergillus, other fungi, and particles following the implosion of an adjacent building; to measure the quantity and persistence of airborne fungi and particles at varying distances during a building implosion; and to determine whether manipulating air systems based on the movement of the dust cloud would be an effective strategy for managing the impact of the implosion. DESIGN: Air sampling study. SETTING: A 976-bed teaching hospital in Baltimore, Maryland. METHODS: Single-stage impactors and particle counters were placed at outdoor sites 100, 200, and 400 m from the implosion and in five locations in the hospital: two oncology floors, the human immunodeficiency virus unit, the cardiac surgical intensive care unit, and the ophthalmology unit. Air handling systems would operate normally unless the cloud approached the hospital. RESULTS: Wind carried the bulk of the cloud away from the hospital. Aspergillus counts rose more than tenfold at outdoor locations up to 200 m from the implosion, but did not increase at 400 m. Total fungal counts rose more than sixfold at 100 and 200 m and twofold at 400 m. Similar to Aspergillus, particle counts rose several-fold following the implosion at 100 and 200 m, but did not rise at 400 m. No increases in any fungi or particles were measured at indoor locations. CONCLUSION: Reacting to the movement of the cloud was effective, because normal operation of the hospital air handling systems was able to accommodate the modest increase in Aspergillus, other fungi, and particles generated by the implosion. Aspergillus measurements were paralleled by particle counts.

Air Conditioning↗
PubMed1986

Synthesis of nitrosomethylisoamylamine from isoamylamine and sodium nitrite by fungi.

Nitrosomethylisoamylamine (NMIA), a carcinogenic N-nitroso compound was synthesized from isoamylamine (IAA) in a glucose-ammonium nitrate medium after several days' incubation with fungi and subsequent nitrosation with sodium nitrate. The nitrosamine was not produced by control reactions which lacked either IAA or fungi. The yield of NMIA varied with the length of incubation after inoculation with the fungi, and with the concentrations of IAA and NaNO2, the duration of nitrosation, the pH of the medium and the species of the fungi. The optimum conditions for the formation of the nitrosamine are reported.

Amines↗
PubMed1994

Phylogenetic spectrum of fungi that are pathogenic to humans.

Recent phylogenetic studies based on ribosomal RNA sequences have confirmed that the organisms traditionally treated as fungi include those that have evolved from several different lines (multiphyletic organisms), as has been suspected. Even organisms causing disease in humans represent at least two evolutional lines. Pythium insidiosum and Prototheca species are both believed to have evolved from one line, while the rest of the pathogens have evolved from another line. P. insidiosum is more closely related to red algae and diatoms than to fungi. Prototheca species, as has been previously postulated, are closer to blue-green algae and plants than to fungi. Pythiosis and protothecosis, however, will still be dealt with by medical mycologists because of the morphological and in vivo staining characteristics of the causative organisms. Molecular genetic studies have revealed that Pneumocystis carinii can best be categorized as a fungus, although questions regarding its fungal status may remain unanswered until additional information becomes available on its life cycle, nuclear division, cell-wall chemistry, nutritional uptake pattern, and lysine biosynthetic pathway as well as the ultrastructural characteristics of its cellular components such as the Golgi complex. The phylogeny of the agents of lobomycosis and rhinosporidiosis, although they are treated as fungi, remains unknown. Although there is no in vitro culture system for Loboa loboi and Rhinosporidium seeberi at present, a molecular approach would allow us to reveal their phylogenetic relationship, and we can hope that such attempts are forthcoming.

DNA, Fungal↗
PubMed1996

Identification of dematiaceous fungi and their role in human disease.

Phaeohyphomycosis is the disease caused by the dematiaceous hyphomycetes or those fungi that are darkly pigmented because of the presence of melanin in their cell wall. Infections with these fungi may remain localized at the site of traumatic inoculation or within the sinuses or may become disseminated. As these fungi pose a significant problem to both the clinician who must treat the patient and the laboratorian faced with identification of the organism, a selection of representative organisms was presented at the meeting entitled "Focus on Fungus Infections." These fungi often infect patients who are not overtly immunocompromised and therefore may be encountered in a wide variety of cases.

Animals↗
PubMed1999

Transposition of the retrotransposon MAGGY in heterologous species of filamentous fungi.

MAGGY is a gypsy-like LTR retrotransposon isolated from the blast fungus Pyricularia grisea (teleomorph, Magnaporthe grisea). We examined transposition of MAGGY in three P. grisea isolates (wheat, finger millet, and crabgrass pathogen), which did not originally possess a MAGGY element, and in two heterologous species of filamentous fungi, Colletotrichum lagenarium and P. zingiberi. Genomic Southern analysis of MAGGY transformants suggested that transposition of MAGGY occurred in all filamentous fungi tested. In contrast, no transposition was observed in any transformants with a modified MAGGY containing a 513-bp deletion in the reverse transcriptase domain. When a MAGGY derivative carrying an artificial intron was introduced into the wheat isolate of P. grisea and C. lagenarium, loss of the intron was observed. These results showed that MAGGY can undergo autonomous RNA-mediated transposition in heterologous filamentous fungi. The frequency of transposition differed among fungal species. MAGGY transposed actively in the wheat isolate of P. grisea and P. zingiberi, but transposition in C. lagenarium appeared to be rare. This is the first report that demonstrates active transposition of a fungal transposable element in heterologous hosts. Possible usage of MAGGY as a genetic tagging tool in filamentous fungi is discussed.

Blotting, Southern↗
PubMed1991

Opportunistic and pathogenic fungi.

The number of fungal species reported to cause disease in man is increasing rapidly. Very few of these fungi are capable of infecting a normal host. Important progress has been achieved in an understanding of fungal pathogenicity including the mechanisms of adherence to host tissues, penetration of tissues, multiplication within the host, and the interaction of fungal cells with host effector cells. In addition to the increase in infections by opportunistic and pathogenic fungi in compromised patients, caused by Candida spp., Aspergillus spp., Cryptococcus neoformans, Histoplasma capsulatum and Coccidioides immitis, many fungi that occur as saprophytes in the environment and which had previously been considered to be nonpathogenic are now being encountered as causes of human infection. The advent of these unusual infections has led to reappraisal of the diagnostic tests involved in the investigation of fungal infections and has had important implications for the choice of treatment. Many of these fungi have a similar tissue appearance and the organisms must be isolated and identified to ensure that the most appropriate treatment is given.

Animals↗
PubMed1995

Phylogenetic analysis of the 90 kD heat shock family of protein sequences and an examination of the relationship among animals, plants, and fungi species.

The heat shock protein (Hsp) sequences, because of their ubiquity and high degree of conservation, provide useful models for phylogenetic analysis. In this paper I have carried out a global alignment of all available sequences (a total of 31) for the 90-kD heat shock protein (Hsp90) family. The minimum amino acid identity that is seen between presently known Hsp90 homologs is about 40% over the entire length, indicating that it is a highly conserved protein. Based on the alignment, a number of signature sequences that either are distinctive of the Hsp90 family or that distinguish between the cytosolic and the endoplasmic reticular forms of Hsp90 have been identified. Detailed phylogenetic analyses based on Hsp90 sequences reported here strongly indicate that the cytosolic and the endoplasmic reticulum (ER) resident forms of Hsp90 constitute paralogous gene families which arose by a gene duplication event that took place very early in the evolution of eukaryotic cells. A minimum of two additional gene duplication events, which took place at a later time, are required to explain the presence of two different forms of Hsp90 that are found in fungi and vertebrate species. In a consensus neighbor-joining bootstrap tree based on Hsp90 sequences, plants and animals species grouped together 989 times of 1,000 (a highly significant score), indicating a closer relationship between them as compared to fungi. A closer affiliation of plant and animal species was also observed in the maximum-parsimony tree, although the relationship was not significantly supported by this method. A survey of the recent literature on this subject indicates that depending on the protein sequence and the methods of phylogenetic analysis, the animal species are indicated as closer relatives to either plants or fungi with significant statistical support for both topologies. Thus the relationship among the animal, plant, and fungi kingdoms remains an unresolved issue at the present time.

Amino Acid Sequence↗
PubMed1995

A simple method for detecting subcutaneous phaeohyphomycosis with light-colored fungi. A study of eight cases.

Eight cases of subcutaneous phaeohyphomycosis are presented. A lack or scarcity of fungal pigmentation was seen in six of these cases. The simple act of lowering the microscope condenser makes these uncolored fungi easily visible. The thick hyaline capsule becomes refringent, presenting single oval or round cells and septated hyphae, which can be short or elongated, branched or unbranched, and irregularly swollen. The dematiacious character of the fungi is confirmed by using the Fontana-Masson stain. The fungi are not visible under polarized light. Our studies point to the probability of subcutaneous phaeohyphomycosis being a widespread tropical disease in Brazil. However, it is rarely diagnosed because of the common practice of fixing tissue specimens without prior culturing and the difficulties of detecting the fungal forms using customary microscopic procedures, especially when the fungi have a lack of pigment and are hard to detect by hematoxylin and eosin stain.

Adult↗
PubMed2003

Melanin and fungi.

PURPOSE OF REVIEW: Production of melanin has been associated with virulence in diverse microorganisms. Melanization of fungi has been noted for many years in predominantly subcutaneous infections such as chromoblastomycosis and more recently most extensively studied in a yeast causing systemic infection, Cryptococcus neoformans. Pigmented fungi are increasingly important human pathogens and currently available antifungals are often sub-optimal for serious infections. This review focuses on recent publications on melanin in fungi with particular reference to the role of melanin in virulence, protection against antifungal drugs, and promoting survival in the environment. RECENT FINDINGS: Inhibition of melanin production by C. neoformans can prolong survival of lethally infected mice. In contrast, melanin in C. neoformans and Histoplasma capsulatum yeast cells can bind amphotericin B and caspofungin, thereby reducing the fungicidal affects of these drugs. H. capsulatum and Paracoccidioides brasiliensis have only recently been shown to produce melanin in vitro and during infection. Additionally, melanin derived from melanized C. neoformans yeast and Aspergillus niger conidia can activate complement, which may modify immune responses to infection. Studies on C. neoformans laccase have revealed that the enzyme is located on the cell wall, which may allow for interactions with the host. Melanization reduces the susceptibility of C. neoformans to enzymatic degradation and toxicity from a heavy metal, which may afford protection to the fungus against similar insults in the environment. SUMMARY: Melanin has been referred to as 'fungal armor' due to the ability of the polymer to protect microorganisms against a broad range of toxic insults. Recent publications continue to reveal important contributions of melanin to survival of fungi in the environment and during infection.

Antifungal Agents↗
PubMed2001

Detection of fungi in the nasal mucosa using polymerase chain reaction.

HYPOTHESIS: Fungi have been increasingly recognized as important pathogens in sinusitis. However, detection of fungus with conventional culture techniques is insensitive and unreliable. Polymerase chain reaction (PCR) is an exquisitely sensitive assay that can detect the DNA of 10 or less fungal elements. The aim of this study was to compare the sensitivity of conventional culture techniques using PCR analysis. METHODS: Nasal swabs and DNA samples were collected from the nasal cavities of control subjects and patients with chronic sinusitis. Fungal-specific PCR analysis and standard cultures were performed on every sample. chi2 analysis was used to test for statistical differences between groups. RESULTS: PCR analysis detected fungal DNA in 42% and 40% of control subjects and patients with chronic sinusitis while standard cultures were positive in 7% and 0%, respectively. There was no statistically significant difference in the prevalence of fungi in the normal volunteers and patients with chronic rhinosinusitis. CONCLUSION: PCR is significantly more sensitive than nasal swab cultures in detecting the presence of fungi in nasal mucosa. In addition, our study suggests that the presence of fungi alone is insufficient to implicate it as the pathogen in chronic sinusitis.

Chronic Disease↗
PubMed2002

In situ hybridization for the identification of filamentous fungi in tissue section.

Identification of fungi in tissue sections can be difficult because of limited biopsy tissue with only a few organisms present, or mycelial elements may be the only forms present, rendering common organism types indistinguishable from one another. In situ hybridization may assist in the rapid and accurate identification of such fungi. In this study, DNA probes were directed against the 5S or 18S ribosomal RNA sequences of three groups of fungi with a high degree of specificity for each. Two of the three, Aspergillus and Zygomycetes species, are usually seen in tissue purely in their hyphal forms. The third, Candida species is seen less commonly as predominantly mycelial elements. Probes were tested on 61 formalin-fixed, paraffin-embedded tissue specimens, each with culture-proven involvement by one of these organisms (Candida species, n = 21; Aspergillus species, n = 27; Zygomycetes, n = 13). Accuracy of both in situ hybridization (ISH) and morphology, based on the examination of Grocott methanamine silver (GMS)- and periodic acid-Schiff (PAS)-stained slides, was compared with culture. The results showed that morphologic examination (GMS and PAS) showed a slightly greater sensitivity in detecting the presence of fungi (98%) compared with in situ hybridization (95%). DNA probes, however, were more accurate in correctly identifying those organisms present. Although ISH specific probes showed 97% positive predictive value (PPV), examination of GMS-and PAS-stained slides had an 86% PPV when compared with culture-based identification methods. These results show that ISH, directed against ribosomal RNA, provides a rapid and accurate technique for the identification of mycelial fungal organisms in histologic tissue sections. Its primary use lies in the ability to accurately distinguish between organisms that have similar or identical morphologic features by light microscopy.

DNA Probes↗
PubMed2002

Production of a monoclonal antibody specific to the genus Trichoderma and closely related fungi, and its use to detect Trichoderma spp. in naturally infested composts.

Studies of the interactions between hyperparasitic fungi and their hosts are severely hampered by the absence of methods that allow the unambiguous identification of individual genera in complex environments that contain mixed populations of fungi, such as soil or compost. This study details the development of a monoclonal antibody (MF2) that allows the detection and recovery of Trichoderma spp. in naturally infested composts, and the visualization of hyperparasitic strains of Trichoderma during antagonistic interactions with their hosts. Murine monoclonal antibody MF2, of immunoglobulin class M (IgM), was raised against a protein epitope of a glycoprotein antigen(s) specific for species of the genus Trichoderma and for the closely related fungi Gliocladium viride, Hypomyces chrysospermus, Sphaerostilbella spp. and Hypocrea spp. MF2 did not react with antigens from Gliocladium catenulatum, Gliocladium roseum, Nectria ochroleuca and Clonostachys spp., nor with a range of unrelated soil- and compost-borne fungi. Extracellular production of the MF2 antigen was constitutive. Western-blotting analysis showed that MF2 bound to a ladder of proteins with apparent molecular masses in the range 35-200 kDa. Immunofluorescence studies showed that MF2 bound strongly to the cell walls of hyphae and phialides and the intercalary and terminal chlamydospores of Trichoderma spp., whereas immunogold electron microscopy revealed strong binding of MF2 to the cell walls and septa of hyphae and to the cell walls of phialoconidia. In immunofluorescence studies of dual cultures of Trichoderma and Rhizoctonia solani, only the cell walls of the hyperparasite, which coiled around the host, were stained by MF2. The specificity of MF2 enabled the development of a combined baiting-ELISA technique for the detection of Trichoderma spp. in naturally infested composts. The specificity of this technique was confirmed by phylogenetic analysis based on sequences of the ITS1-5.8S-ITS2 rRNA-encoding regions of the isolates.

Antibodies, Fungal↗
PubMed2006

Fast and reliable PCR/sequencing/RFLP assay for identification of fungi in onychomycoses.

Fusarium spp. and other non-dermatophyte fungi are repeatedly isolated from abnormal nails. To investigate whether these fungi are the aetiological agents of infection or simply transient contaminants, a PCR/sequencing/RFLP assay was developed for direct and routine identification of the infecting fungi in onychomycosis. Fungal DNA was readily extracted using a commercial kit after dissolving nail fragments in a Na2S solution. Amplification of part of the 28S rDNA by PCR was performed with universal primers and the fungal species were identified by sequencing. The PCR/sequencing results were comparable with microbiological identification from the same nail sample. In addition to dermatophytes, Fusarium spp. and other less frequently isolated non-dermatophyte fungi were identified as single fungal agents in onychomycosis. Moreover, mixed infections were clearly demonstrated in 10% of cases by RFLP analysis of PCR products. Identification of infectious agents could be obtained in 2 days, whilst results from fungal cultures take 1-3 weeks. Rapid and reliable molecular identification of the infectious fungus expedites the choice of appropriate antifungal therapy, thereby improving the cure rate of onychomycosis.

DNA, Fungal↗
PubMed2007

The first fifty microarray studies in filamentous fungi.

Microarray studies have examined global gene expression in over 20 species of filamentous fungi encompassing a wide variety of research areas. The majority have addressed aspects of metabolism or pathogenicity. Metabolic studies have revealed important differences in the transcriptional regulation of genes for primary metabolic pathways between filamentous fungi and yeast. Transcriptional profiles for genes involved in secondary metabolism have also been established. Genes required for the biosynthesis of both useful and detrimental secondary metabolites have been identified. Due to the economic, ecological and medical implications, it is not surprising that many studies have used microarray analysis to examine gene expression in pathogenic filamentous fungi. Genes involved in various stages of pathogenicity have been identified, including those thought to be important for adaptation to the host environment. While most of the studies have simulated pathogenic conditions in vitro, a small number have also reported fungal gene expression within their plant hosts. This review summarizes the first 50 microarray studies in filamentous fungi and highlights areas for future investigation.

Adaptation, Physiological↗