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At least 19 recordsLinked to original sources
PubMed2005

Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR.

The detection of fungal pathogens in clinical samples by PCR requires the use of extraction methods that efficiently lyse fungal cells and recover DNA suitable for amplification. We used quantitative PCR assays to measure the recovery of DNA from two important fungal pathogens subjected to six DNA extraction methods. Aspergillus fumigatus conidia or Candida albicans yeast cells were added to bronchoalveolar lavage fluid and subjected to DNA extraction in order to assess the recovery of DNA from a defined number of fungal propagules. In order to simulate hyphal growth in tissue, Aspergillus fumigatus conidia were allowed to form mycelia in tissue culture media and then harvested for DNA extraction. Differences among the DNA yields from the six extraction methods were highly significant (P<0.0001) in each of the three experimental systems. An extraction method based on enzymatic lysis of fungal cell walls (yeast cell lysis plus the use of GNOME kits) produced high levels of fungal DNA with Candida albicans but low levels of fungal DNA with Aspergillus fumigatus conidia or hyphae. Extraction methods employing mechanical agitation with beads produced the highest yields with Aspergillus hyphae. The Master Pure yeast method produced high levels of DNA from C. albicans but only moderate yields from A. fumigatus. A reagent from one extraction method was contaminated with fungal DNA, including DNA from Aspergillus and Candida species. In conclusion, the six extraction methods produce markedly differing yields of fungal DNA and thus can significantly affect the results of fungal PCR assays. No single extraction method was optimal for all organisms.

Aspergillosis↗
PubMedDate not supplied

Fungal DNA is present in tissue specimens of patients with chronic rhinosinusitis.

BACKGROUND: It has been postulated that fungal organisms might represent the immunologic target initiating and maintaining the disease process in patients with chronic rhinosinusitis (CRS). The presence of fungi in nasal mucus has been established by different groups, but so far it has not been shown how the immune system could even recognize such extramucosal--extracorporal--fungal targets. The aim of this study was to determine whether fungal DNA is present in tissue specimens taken from patients with polypoid CRS. METHODS: Twenty-seven surgical specimens were collected from patients suffering from CRS. Fifteen surgical specimens from healthy ethmoidal mucosa served as controls. A second set of controls consisted of five surgical specimens of acoustic neuroma, which were included to rule out contamination within the protocol. All paranasal tissue samples were treated and rinsed carefully with a solution of Dithiothreitol to digest any nasal mucus and ensure that only tissue was examined. A highly sensitive two-step polymerase chain reaction (PCR) was applied to detect fungal DNA, using one universal primer for unspecific detection of fungal DNA and a second primer pair specific for Alternaria. RESULTS: Fungal DNA was detected in all 27 CRS specimens equally with both PCR primers. Controls from healthy paranasal mucosa were positive using the panfungal primers in 10 of 15 cases but were all negative for Alternaria DNA. PCR was negative for fungal DNA in all five neuroma specimens. CONCLUSIONS: Fungal DNA can be detected within sinonasal tissue specimens of patients suffering from CRS. These findings need to be discussed with respect to the proposed hypothesis of the immune system recognizing extramucosal organisms and initiating an immune response in sensitized patients.

Adolescent↗
PubMed2004

Quantitative analysis of fungal DNA in chronic rhinosinusitis.

OBJECTIVES/HYPOTHESIS: Fungi have been recognized as important pathogens in sinusitis; however, they are equally present in patients with and without sinusitis. The authors postulated that the quantity of fungal DNA in the nose is determinant of disease, is greater in patients with chronic rhinosinusitis, and is directly correlated to their quality of life. STUDY DESIGN: Prospective recruitment of patients with chronic rhinosinusitis. METHODS: Objective quality of life data were collected using three validated questionnaires: the Sinonasal Outcomes Test (SNOT-20), Medical Outcomes Short-Form 36 Survey (SF-36), and Guy Marks Asthma Questionnaire (GMAQ). Endoscopically guided middle meatus mucosal samples were collected from patients with chronic rhinosinusitis and normal control subjects. Fungal-specific polymerase chain reaction was performed on each sample. Every fungal-positive sample underwent fungal-specific quantitative polymerase chain reaction analysis. Statistical analysis was used to correlate fungal DNA quantities with outcomes indices between groups. RESULTS: Patients with chronic rhinosinusitis had a mean SNOT-20 index of 32.0 as compared with a SNOT-20 index of 17.3 (P <.01) in the normal control subjects. There were no statistical differences between the groups' indices for the SF-36 or GMAQ outcomes questionnaires. Four of 19 (21.1%) patients with chronic rhinosinusitis and 7 of 19 (36.8%) normal control subjects had positive findings for fungal DNA using polymerase chain reaction. The median relative quantity of fungal DNA to human DNA for chronic rhinosinusitis and control samples was identical (0.13) using quantitative polymerase chain reaction. CONCLUSION: The quantity of fungal DNA in the middle meatus did not differ in patients with and without chronic rhinosinusitis and was not correlated with quality of life outcomes. Therefore, the quantity of fungi does not explain pathogenicity in patients with chronic rhinosinusitis. However, because of small sample size, the study must be replicated in a larger patient population.

Chronic Disease↗
PubMed2004

Rapid identification and differentiation of fungal DNA in dermatological specimens by LightCycler PCR.

The aim was to develop a LightCycler PCR method for the rapid detection and differentiation of fungal DNA in dermatological specimens such as skin scales and skin swabs. LightCycler PCR assays were established for seven primer sets specific for fungal DNA. For each primer set LightCycler melting points were defined by amplification of DNA from 21 fungi and sensitivity was determined by amplification of serial dilutions of fungal DNA. A protocol was established that allows detection and differentiation of mould and yeast DNA with one highly sensitive PCR reaction by assessment of LightCycler melting points. Two subsequent LightCycler PCR reactions and one RFLP reaction allowed the differentiation of dermatophytes and non-dermatophyte moulds and the subclassification of yeasts. Analysis of clinical samples from 38 patients with fungal skin diseases provided conclusive new diagnostic information in 9/38 cases (23.7 %) by this PCR protocol that was not equally provided by direct microscopy and mycological culture. Thus the LightCycler PCR protocol established here represents a rapid diagnostic tool that aids in the diagnosis of fungal skin disease in a substantial number of patients.

DNA, Fungal↗
PubMed2003

A simple protocol for isolation of fungal DNA.

Genomic DNA was isolated from as little as 2 mg dry biomass of Magnaporthe grisea by microwave treatment within 30 s. The quantity of DNA was good enough for PCR analysis and Dot blot hybridization. This technique can be used for various studies, such as DNA fingerprinting to study the population structure of the phytopathogen in different regions, and for a quick screening of M. grisea transformants.

DNA, Fungal↗
PubMed2005

Fungal DNA challenge in human STR typing of bone samples.

The present study focuses on possible cross-reaction of fungal DNA with human STR primers that may affect subsequent forensic DNA analysis of forensic samples. Specificity of human STR markers namely HUMAMEL, HUMCSF1PO, D8S306, HUMTH01, HUMvWA, HUMFES/FPS, HUMF13A01, HUMDHFRP2, HUMFGA and HUMTPOX was tested using DNA of 24 different filamentous fungal isolates obtained from exhumed bone samples. The specificity of these ten STR markers for human DNA was demonstrated. Presence of non-human DNA in five bone samples analyzed did not alter scoring of detected alleles. Notably, amplification was inhibited in the presence of a high proportion of fungal DNA compared to human DNA (1000 ng: 1 ng) in DNA mixture experiments. The results of the present study underscore the importance of carefully analyzing the presence of non-human biological contaminants that may affect DNA typing of environmentally challenged forensic samples to avoid spurious data interpretation.

Bone and Bones↗
PubMed1999

Rapid extraction of fungal DNA from clinical samples for PCR amplification.

A hexadecyltrimethylammonium bromide (CTAB) method for isolating fungal DNA from clinical samples, suitable for PCR amplification is described. Yeast and filamentous fungi DNA from clinical samples was amplified with primers complementary to the genes coding for rRNA, amplifying a 105 bp fragment and internal transcribed spacer primers amplifying fragments between 242 and 622 bp. The level of sensitivity was 10 +/- 5 yeast and 28 Aspergillus fumigatus CFU ml-1 of biological fluid.

Blood↗
PubMed1999

Identification of contaminating fungal DNA sequences in Zymolyase.

When different preparations of Zymolyase were included in the pretreatment protocol of a panfungal PCR assay using a primer system for the 18S rRNA gene, an amplification product occurred in negative controls. The amplified fragment showed 100.0% sequence identity to the Saccharomyces sensu stricto complex and Kluyveromyces lodderae. Lyticase, lysing enzymes, and proteinase K appeared to be free from fungal DNA.

DNA Primers↗
PubMed2004

The fungal DNA examination is useful as a sensitive parameter for the initiation and the quit of antifungal therapy in immunocompromised pediatric patients after surgery.

The purpose of this study is whether the fungal deoxyribonucleic acid (DNA) examination is useful as a sensitive parameter for pediatric surgical patients with mycosis. The eleven episodes of five cases (4 cases; progressive liver disease after biliary atresia operation, 1 case; short bowel syndrome and long term total parenteral nutrition with megacystis microcolon intestinal hypoperistalsis syndrome) with mycosis were divided into two groups according to the difference of therapeutic protocols. The sensitivity of fungal DNA examination, serum Candida antigen level, plasma beta-D glucan level, and blood culture were evaluated at the onset of infection and at the quit of antifungal medication under the protocols respectively. The duration of medication and the medication free interval in two groups were compared. The 6 episodes (3 cases) were diagnosed and treated under the protocol not including fungal DNA examination, while the 5 episodes (2 cases) under the protocol including fungal DNA examination. The occurrence rate was not significant. The sensitivity of fungal DNA examination was complete, but others were not. Using the fungal DNA examination, the duration of medication became significantly short. We conclude that the fungal DNA examination could be a sensitive parameter not only to start but to quit antifungal medication in pediatric patients with mycosis.

Antifungal Agents↗
PubMed2006

Broad-range bacterial and fungal DNA amplification on vitreous humor from suspected endophthalmitis patients.

BACKGROUND: Prompt and accurate diagnosis of infectious endophthalmitis is crucial for rapid and effective treatment. By identifying whether the causative pathogen is bacterial or fungal, a rational approach for the use of antibacterials or corticosteroids, respectively, can be followed. AIM: To assess the clinical utility of broad-range bacterial and fungal DNA amplification in the detection of endophthalmitis (postoperative, posttraumatic, and endogenous). METHODS: In a prospective study, vitreous humor samples from 70 patients with the clinical diagnosis of presumed endophthalmitis, and from 30 patients undergoing surgery for non-infectious causes, were subjected to routine microbiologic and molecular investigation. DNA extracted from a 50 microL sample was amplified by primers targeting the conserved 16S and 18S ribosomal RNA gene sequences of bacteria and fungi, respectively. Reagents for bacterial DNA amplification were decontaminated of endogenous DNA using 8-methoxypsoralen and long wave UV treatment. RESULTS AND DISCUSSION: A total of 35 specimens were positive for bacteria or fungi by culture. Of these, Gram-positive organisms were isolated in 19 specimens, Gram-negative organisms in 13 specimens and fungi in 3 specimens. Pseudomonas species, coagulase-negative Staphylococcus, and Streptococcus species were the main etiological agents isolated. Bacterial DNA amplification resulted in 49 positive specimens, compared with 32 positive specimens by culture; and fungal DNA amplification resulted in 11 positive specimens, compared with 3 positive specimens by culture. All control specimens were negative for both culture and DNA amplification. CONCLUSION: DNA extracted using a single-extraction protocol from 50 microL of vitreous humor and amplified with broad-range bacterial and fungal primers will enable the rapid differentiation (within 14 hours) between bacterial and fungal endophthalmitis and allow tailoring of therapy to individual patients.

Bacteria↗
PubMedDate not supplied

Evaluation of fungal DNA sequence using polymerase chain reaction in infection-free vitreous humor.

We evaluated the vitreous bodies from 14 clinically nonfungal infectious eyes to establish a baseline for the presence of a fungal genome using polymerase chain reaction (PCR). The vitreous samples were all PCR negative, except for one eye with chronic uveitis. Blood samples examined were all PCR negative. We concluded that fungal DNA sequences could not be amplified in vitreous specimens obtained from patients with clinically diagnosed proliferative diabetic retinopathy, macular hole, or epiretinal membrane.

Adult↗
PubMed2005

Use of magnetic beads to extract fungal DNA.

Authors compare two methods of extracting DNA from different fungi: the classic method with phenol/chloroform (P/C) and that with magnetic beads. Both were tested on Candida albicans and Cryptococcus neoformans var. neoformans, belonging to the yeast group and Microsporum canis, M. gypseum, Trichophyton rubrum, T. interdigitale, T. ajelloi, Epidermophyton floccosum, belonging to the dermatophytes group. Extraction products underwent polymerase chain reaction (PCR) fingerprinting with the appropriate primers to point out any disagreement in the genomic profiles. After having determined that the genomic profiles obtained from the DNA extracted from the same strain with the two methods correspond perfectly, the authors concluded that the extraction method with magnetic beads from fungal cells is simpler and quicker than with P/C extraction, greatly facilitating the obtainment of fungal DNA.

Arthrodermataceae↗
PubMed1982

Photochemical interaction of dictamnine, a furoquinoline alkaloid, with fungal DNA.

The furoquinoline alkaloid dictamnine has been shown to provoke lethal damage to filamentous fungi in near ultraviolet light. The phototoxicity was more pronounced against Mucor hiemalis and Mucor ramannianus than against Fusarium graminearum and Penicillium italicum. In vitro, labeled dictamnine was shown to form covalent monoadducts with purified DNA from M. hiemalis in the presence of long-wave ultraviolet light. Addition of [3H]dictamnine to cultures of the same organism showed photobinding with the fungal DNA in vivo. These results support the suggestion made earlier, on the basis of in vitro experiments with calf thymus DNA, that DNA represents a major cellular target in vivo for the phototoxicity of the alkaloid.

Alkaloids↗