Assessment of the significance of respiratory culture of Aspergillus in the non-neutropenic patient. A critique of published diagnostic criteria.

Assessment of the significance of isolation of Aspergillus sp. from respiratory culture in patients who are not neutropenic is a continuing problem in respiratory medicine. In recent years a number of criteria for defining patients with invasive or chronic pulmonary aspergillosis in this group have been proposed. This study sought to assess the value of three sets of these criteria in distinguishing between colonisation and aspergillosis requiring therapy when applied retrospectively to 121 patients with positive sputum or BAL culture for Aspergillus sp. Two patients (1.6%) were identified as having proven or probable aspergillosis by the EORTC criteria, two different patients fulfilled the criteria for invasive aspergillosis in the 62 patients with chronic obstructive pulmonary disease (3.2%), and yet another two different patients met the criteria for chronic pulmonary aspergillosis (1.6%). It is suggested that difficulties in the application of some of these criteria may prevent the accurate diagnosis of aspergillosis in the non-neutropenic patient setting.

Isolation of the fungus Geosmithia argillacea in sputum of people with cystic fibrosis.

We report the repeated isolation of the fungus Geosmithia argillacea from sputum samples of people with cystic fibrosis. Identification was based on morphology and DNA sequence analysis. Isolation of G. argillacea did not appear to be associated with clinical deterioration. The pathogenic potential of G. argillacea is discussed.

Use of multilocus sequence typing for the investigation of colonisation by Candida albicans in intensive care unit patients.

A prospective study was performed to determine the prevalence of candidal colonisation on the general intensive care unit at a large teaching hospital. Colonisation with Candida spp. was found to be common, occurring in 79% of patients on the unit. C. albicans was the commonest species, colonising 64% of patients, followed by C. glabrata (18%) and C. parapsilosis (14%). Most of the members of staff tested carried Candida spp. at some point, although carriage appeared to be transient. C. parapsilosis was the most commonly isolated species from staff hands, whereas C. albicans was the most commonly isolated species from the mouth. The molecular epidemiology of C. albicans was investigated using Ca3 typing and multilocus sequence typing (MLST). MLST proved to be a reproducible typing method and a useful tool for the investigation of the molecular epidemiology of C. albicans. The results of the molecular typing provided evidence for the presence of an endemic strain on the unit, which was isolated repeatedly from patients and staff. This finding suggests horizontal transmission of C. albicans on the unit though it may also reflect the relative frequency of C. albicans strain types colonising patients on admission. This study has important implications for the epidemiology of systemic candidal infections.

Cutaneous infection with an Alternaria sp. in an immunocompetent host.

We report a case of cutaneous alternariosis in an immunocompetent 85-year-old man. Identification of the causative organism in this patient was achieved by 18S rRNA sequencing. A 6-month course of itraconazole led to a good clinical response.

Hydrothorax in association with Scopulariopsis brumptii in an AIDS patient in Chennai, India.

Over the past decade, an increasing number of opportunistic fungal infections have been reported in immunocompromised subjects. Microascus spp. and their anamorphs Scopulariopsis spp. have been recovered from a wide geographical range. We report a case of Scopulariopsis brumptii in a 27-year-old man with AIDS presenting with breathlessness, pericardial effusion and hydrothorax in Chennai, southern India, in February 2007. Because of respiratory arrest, the patient was intubated. However, the patient developed obstructive shock and died due to cardiac dysfunctions. This report underlines the need for a direct, intensive approach to investigations in immunocompromised patients, especially those with AIDS.

Inconsistent identification of pit bull-type dogs by shelter staff.

Shelter staff and veterinarians routinely make subjective dog breed identification based on appearance, but their accuracy regarding pit bull-type breeds is unknown. The purpose of this study was to measure agreement among shelter staff in assigning pit bull-type breed designations to shelter dogs and to compare breed assignments with DNA breed signatures. In this prospective cross-sectional study, four staff members at each of four different shelters recorded their suspected breed(s) for 30 dogs; there was a total of 16 breed assessors and 120 dogs. The terms American pit bull terrier, American Staffordshire terrier, Staffordshire bull terrier, pit bull, and their mixes were included in the study definition of 'pit bull-type breeds.' Using visual identification only, the median inter-observer agreements and kappa values in pair-wise comparisons of each of the staff breed assignments for pit bull-type breed vs. not pit bull-type breed ranged from 76% to 83% and from 0.44 to 0.52 (moderate agreement), respectively. Whole blood was submitted to a commercial DNA testing laboratory for breed identification. Whereas DNA breed signatures identified only 25 dogs (21%) as pit bull-type, shelter staff collectively identified 62 (52%) dogs as pit bull-type. Agreement between visual and DNA-based breed assignments varied among individuals, with sensitivity for pit bull-type identification ranging from 33% to 75% and specificity ranging from 52% to 100%. The median kappa value for inter-observer agreement with DNA results at each shelter ranged from 0.1 to 0.48 (poor to moderate). Lack of consistency among shelter staff indicated that visual identification of pit bull-type dogs was unreliable.

Detection of Aspergillus fumigatus PCR products by a microtitre plate based DNA hybridisation assay.

AIMS: To develop a DNA based plate hybridisation assay for the detection of polymerase chain reaction (PCR) products amplified from Aspergillus fumigatus DNA; and to determine the sensitivity of this technique and compare it with Southern blotting. METHODS: A half-log dilution series of DNA extracted from A fumigatus was amplified with specific primers, one of which was 5' end labelled with biotin. PCR products were subsequently detected by agarose gel electrophoresis, Southern blotting, and binding of the products to a streptavidin coated microtitre well, followed by non-radioactive colorimetric detection. Amplification was carried out 10 times for each DNA dilution and a plot of initial DNA concentration against signal intensity was made. RESULTS: A DNA concentration of 1.5 pg could be detected by agarose gel electrophoresis and Southern blotting with a non-radioactively labelled aspergillus specific probe; 1.5 pg was detectable by streptavidin binding of the PCR products to a microtitre plate. The signal from the microtitre plate detection was proportional to the amount of DNA in the PCR reaction on a log-log scale between 100 and 1 pg of DNA. CONCLUSIONS: A DNA based plate hybridisation assay for the detection of A fumigatus PCR products is as sensitive as Southern blotting. However, results are obtained in three hours rather than the three days required for agarose gel electrophoresis, blotting, hybridisation, and detection.

Cerebral phaeohyphomycosis caused by Fonsecaea monophora.

We report a case of cerebral phaeohyphomycosis in a 53-year-old immunocompetent diabetic male, caused by Fonsecaea monophora. Computerized tomography of the brain revealed an abscess, which yielded F. monophora in pure culture. The patient's condition deteriorated on treatment with voriconazole and 5-fluorocytosine, and improved subsequently with high-dose itraconazole. The genus Fonsecaea has recently been revised and the new species F. monophora generated from molecular analysis of isolates, most of which were originally identified as Fonsecaea pedrosoi. This is the fourth case of cerebral infection known to have been caused by F. monophora, although only the first reported as such. These cases suggest that the clinical potential of F. monophora differs from that of F. pedrosoi, one of the main agents of chromoblastomycosis, with F. monophora being predominantly neurotropic in the human host.

Induced chromosome rearrangements and morphologic variation in Candida albicans.

We have isolated a mutant of Candida albicans that switches between colony morphologies at high frequencies in a strain with several genetic markers. This strain, 1183, has an altered karyotype with two extra chromosomes. The 1183 karyotype is unstable upon passage. Using DNA transformation with the URA3 gene flanked by sequences from the C. albicans repeat sequence 27A, we have marked individual chromosomes of 1183 and 1161, a related smooth, stable strain. Many transformants contained one or more extra chromosomes, ranging in size from 150 kb to 2.1 Mb. Most were less than 800 kb and appeared to be fragments of a single chromosome. All fragments tested derive from one of the two smallest chromosomes. Six of 13 fragments contained the URA3 gene. In some cases, URA3 was located at the end of a fragment with adjacent telomere repeats. The integrated copy of URA3 was unstable in some 1183 transformants. Our results suggest that 1183 has a mutation affecting genomic stability. A connection between karyotypic changes and morphologic variation has been suggested from studies of several C. albicans strains; however, we find that gross karyotypic and morphological changes are separable processes.

Isolation, characterization, and genetic analysis of monosomic, aneuploid mutants of Candida albicans.

A white, prototrophic Candida albicans strain, heterozygous for the ADE2 gene (ade2/ADE2), was treated with the antimitotic agent methyl benzimidazole carbamate, and yielded red, adenine-requiring colonies at a rate of 4 x 10(-3), an order of magnitude higher than the spontaneous rate of Ade- colony formation. These red Ade- colonies were small, growing at approximately half the rate of the parent strain, and gave rise to large red colonies spontaneously. When the chromosomes of the small red colonies were separated by pulsed-field gel electrophoresis, the band hybridizing with the ADE2 gene was diminished in staining intensity by half relative to the parent and large red-colony strains. Restriction fragment-length polymorphism analysis and auxotrophic mutant spectra after mutagenesis suggested that the small red Ade- strains were monosomic aneuploids lacking one of a pair of chromosome homologues, while the large red strains had regained a homologue, presumably via a second non-disjunction event. Parasexual genetic analysis of two of the auxotrophs isolated from a putative aneuploid suggested that both mutations were linked to the ADE2 gene. These experiments suggest that targeted chromosome loss and monosomic, aneuploid strains have the potential to extend the scope of genetic analysis in this diploid, asexual organism.

Cryopreservation of Aspergillus fumigatus stock cultures with a commercial bead system.

A method using beads and storage at -80 degrees C was used to maintain isolates of the pathogenic mould Aspergillus fumigatus. Viability was assessed after 6 and 15 months and all isolates were recovered in culture. This inexpensive technique was found to be an effective and easy method of preserving A. fumigatus and other pathogenic moulds.

Stability of karyotype in serial isolates of Candida albicans from neutropenic patients.

Serial isolates of Candida albicans were obtained from 20 patients with leukemia over periods of up to 8 months. The fingerprinting of these isolates by interrepeat PCR and random amplified polymorphic DNA PCR has been described previously (A. van Belkum, W. Melchers, B. E. de Pauw, S. Scherer, W. Quint, and J. F. Meis, J. Infect. Dis. 169:1062-1070, 1994). Contour-clamped homogeneous electric field gel electrophoresis was used to examine the chromosomes of these isolates. When changes in the karyotype were seen in a series of isolates, additional interrepeat PCR and Southern blotting with a repeat DNA sequence from the 27A family were performed. These two genotyping tools were used to determine if karyotypic changes seen in a series of isolates were due to chromosome rearrangements in a single strain or due to colonization with more than one strain. It was determined that changes in karyotype in a series of strains indicated infection by a new strain.

Species identification and strain differentiation of dermatophyte fungi by analysis of ribosomal-DNA intergenic spacer regions.

Restriction fragment length polymorphisms (RFLPs) identified in the ribosomal-DNA (rDNA) repeat were used for molecular strain differentiation of the dermatophyte fungus Trichophyton rubrum. The polymorphisms were detected by hybridization of EcoRI-digested T. rubrum genomic DNAs with a probe amplified from the small-subunit (18S) rDNA and adjacent internal transcribed spacer (ITS) regions. The rDNA RFLPs mapped to the nontranscribed spacer (NTS) region of the rDNA repeat and appeared similar to those caused by short repetitive sequences in the intergenic spacers of other fungi. Fourteen individual RFLP patterns (DNA types A to N) were recognized among 50 random clinical isolates of T. rubrum. A majority of strains (19 of 50 [38%]) were characterized by one RFLP pattern (DNA type A), and four types (DNA types A to D) accounted for 78% (39 of 50) of all strains. The remaining types (DNA types E to N) were represented by one or two isolates only. A rapid and simple method was also developed for molecular species identification of dermatophyte fungi. The contiguous ITS and 5.8S rDNA regions were amplified from 17 common dermatophyte species by using the universal primers ITS 1 and ITS 4. Digestion of the amplified ITS products with the restriction endonuclease MvaI produced unique and easily identifiable fragment patterns for a majority of species. However, some closely related taxon pairs, such as T. rubrum-T. soudanense and T. quinkeanum-T. schoenlenii could not be distinguished. We conclude that RFLP analysis of the NTS and ITS intergenic regions of the rDNA repeat is a valuable technique both for molecular strain differentiation of T. rubrum and for species identification of common dermatophyte fungi.

Molecular strain typing of Trichophyton rubrum indicates multiple strain involvement in onychomycosis.

BACKGROUND: Trichophyton rubrum is an important cause of onychomycosis. Molecular strain typing methods have recently been developed to address questions of epidemiology and source of relapse following treatment. OBJECTIVES: To determine whether T. rubrum nail infections are caused by one or more strains of this fungus. METHODS: Nail specimens from 10 patients with onychomycosis due to T. rubrum were cultured and five colonies per culture plate were selected for molecular strain typing. DNA was extracted from these isolates and subjected to a polymerase chain reaction-based typing method that analyses variations in numbers of repetitive elements in the non-transcribed spacer region of the ribosomal RNA gene repeats. RESULTS: In six of 10 specimens, there were two or more T. rubrum strain types present. CONCLUSIONS: This preliminary study suggests that in many cases of fungal nail infection by T. rubrum, multiple strains are involved. This has important implications for epidemiological studies and possibly for therapy.

Determination of optimum growth conditions for gliotoxin production by Aspergillus fumigatus and development of a novel method for gliotoxin detection.

Gliotoxin is a toxic metabolite of Aspergillus fumigatus Fresenius and other fungi. It has been suggested that this toxin may play an important role in the pathogenesis of aspergillosis as gliotoxin has immunosuppressive activity both in vitro and in vivo. We have determined the optimum growth conditions for the production of gliotoxin by selected isolates of A. fumigatus using a number of defined media. Gliotoxin was detected by thin layer chromatography and high performance liquid chromatography. The carbohydrate source, concentration of carbohydrate in the growth medium and incubation temperature were all found to influence gliotoxin production. Optimum growth conditions for gliotoxin production in our study were Czapek-Dox broth containing 30% glucose and incubation at 37 degrees C. Most of the gliotoxin was produced after 29 h incubation, during the exponential phase of growth. A novel method for screening large numbers of A. fumigatus isolates for gliotoxin production, which is both quick and easy, has also been developed, based on the ability of gliotoxin to inhibit the adherence of lung fibroblast (L929) cells to plastic microtitre plates.

Strain identification of Trichophyton rubrum by specific amplification of subrepeat elements in the ribosomal DNA nontranscribed spacer.

Trichophyton rubrum is the commonest cause of dermatophytosis of skin and nail tissue. Molecular characterization of the T. rubrum ribosomal DNA nontranscribed-spacer region revealed two novel tandemly repetitive subelements (TRSs): TRS-1, containing a 27-bp palindromic sequence, and TRS-2. Specific amplification of TRS-1 produced strain-characteristic banding patterns (PCR types), with 21 TRS-1 PCR types recognized from 101 clinical isolates. Four simple patterns representing 1 to 4 copies of TRS-1 accounted for 75 (75%) of all 101 strains, whereas more complex patterns were observed for 21 (20%) of the 101 isolates. The copy number of TRS-2 was 0 to 3 repeats per cistron, with a majority of isolates having two copies of this element. Eleven isolates were polymorphic for TRS-2, and in combination, 23 separate PCR types were recognized by amplification of both TRS-1 and TRS-2. The PCR patterns from both elements were stable and reproducible. Elements with homology to TRS-1 were present in three phylogenetically related species, Trichophyton violaceum, Trichophyton gourvilii, and Trichophyton soudanense, but these elements were not identified in other dermatophyte taxa. There was no clear correlation of PCR type with specimen (skin or nail tissue), but certain PCR types appeared to show a bias in geographic distribution. This new method of typing T. rubrum will enable important questions about pathogenesis and epidemiology of this fungus to be addressed.

Microbiological and molecular diagnosis of deep localized cutaneous infection with Trichophyton mentagrophytes.

We describe a healthy young woman with a localized deep dermal infection on the right side of the chest wall. It was caused by the dermatophyte Trichophyton mentagrophytes, and resolved after two pulses of oral itraconazole 200 mg twice daily for 1 week. As cultural and microscopic features did not enable a precise identification of the fungus, molecular investigation was undertaken. Patterns of HaeIII restriction digests of genomic DNA from the culture matched those from Arthroderma incurvata and A. benhamiae, which is the teleomorph of T. mentagrophytes var. mentagrophytes.

An investigation into the pathogenesis of vulvo-vaginal candidosis.

OBJECTIVE: To monitor yeasts isolated from women during and between episodes of recurrent vulvo-vaginal candidosis (VVC) to determine whether vaginal relapse or re-infection occurred. METHODS: Women presenting at the genitourinary medicine clinic with signs and symptoms of VVC were recruited to the study (n = 121). A vaginal washing, high vaginal swab (HVS) and rectal swab were taken and the women treated with a single 500 mg clotrimazole pessary. Women were asked to re-attend after 1, 4, and 12 weeks, or when the VVC recurred, when vaginal washings and HVS were repeated. Candida isolates recovered were strain typed using the Ca3 probe and their similarity assessed. Antifungal susceptibility to fluconazole and clotrimazole were determined. RESULTS: Of the women recruited, 47 completed the study, either returning for four visits or suffering a recurrence during the study period. Of the 22 women who experienced recurrence, the same strain was responsible for the initial and recurrent episode in 17 women. For the remaining five women, four had strain replacement and one had a change of species. None of the isolates recovered from the women demonstrated resistance to either clotrimazole or fluconazole. CONCLUSIONS: Our findings support the theory of vaginal relapse and thus may support the use of more prolonged courses of antifungal therapy initially to increase the chances of eradication of the yeast.

Non-tuberculous mycobacterial lung infection complicated by chronic necrotising pulmonary aspergillosis.

We report four cases of pulmonary mycobacterial disease (three due to Mycobacterium malmoense and one to Myco- bacterium avium intracellulare) complicated by the development of chronic necrotising pulmonary aspergillosis. Difficulties with treatment and the potential benefits of steroids are discussed.