Dermatopathology and the Diagnosis of Fungal Infections.

Diagnosis of superficial/cutaneous fungal infections from skin, hair and nail samples is generally achieved using microscopy and culture in a microbiology laboratory, however, any presentation that is unusual or subcutaneous is sampled by taking a biopsy. Using histological techniques a tissue biopsy enables a pathologist to perform a full examination of the skin structure, detect any inflammatory processes or the presence of an infectious agent or foreign body. Histopathological examination can give a presumptive diagnosis while a culture result is pending, and may provide valuable diagnostic information if culture fails. This review demonstrates how histopathology contributes to the diagnosis of fungal infections from the superficial to the life threatening.

Mammalian Golgi-associated Bicaudal-D2 functions in the dynein-dynactin pathway by interacting with these complexes.

Genetic analysis in Drosophila suggests that Bicaudal-D functions in an essential microtubule-based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. We show here that a mammalian homologue of Bicaudal-D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co-localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein-tagged forms of BICD2 verify its intracellular distribution and co-localization with dynactin, and indicate that the C-terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N-terminal domain of BICD2 disrupts minus-end-directed organelle distribution and this portion of BICD2 co-precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2-dynactin-dynein co-localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein- dynactin interaction on the surface of membranous organelles, by associating with these complexes.

Proteolytic processing and primary structure of Plasmodium falciparum apical membrane antigen-1.

Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a malaria merozoite integral membrane protein that plays an essential but poorly understood role in invasion of host erythrocytes. The PfAMA-1 ectodomain comprises three disulfide-constrained domains, the first of which (domain I) is preceded by an N-terminal prosequence. PfAMA-1 is initially routed to secretory organelles at the apical end of the merozoite, where the 83-kDa precursor (PfAMA-1(83)) is converted to a 66-kDa form (PfAMA-1(66)). At about the time of erythrocyte invasion, PfAMA-1(66) selectively translocates onto the merozoite surface. Here we use direct microsequencing and mass spectrometric peptide mass fingerprinting to characterize in detail the primary structure and proteolytic processing of PfAMA-1. We have determined the site at which processing takes place to convert PfAMA-1(83) to PfAMA-1(66) and have shown that both species possess a completely intact and unmodified transmembrane and cytoplasmic domain. Following relocation to the merozoite surface, PfAMA-1(66) is further proteolytically cleaved at one of two alternative sites, either between domains II and III, or at a membrane-proximal site following domain III. As a result, the bulk of the ectodomain is shed from the parasite surface in the form of two soluble fragments of 44 and 48 kDa. PfAMA-1 is not detectably modified by the addition of N-linked oligosaccharides.

Co-transfer of plasmids in association with conjugative transfer of mupirocin or mupirocin and penicillin resistance in methicillin-resistant Staphylococcus aureus.

Two distinct strains of methicillin-resistant Staphylococcus aureus (MRSA) isolated from patients in a dermatology ward were also resistant to mupirocin. The mupirocin resistance plasmids from both strains were indistinguishable by EcoRI and HindIII restriction digest analysis, except for the presence of genes apparently mediating penicillinase production in some transconjugants. Conjugative transfer of the plasmid mediating mupirocin resistance from one of these strains to a recipient S. aureus was accompanied in some cases by co-transfer of plasmids mediating resistance to tetracycline or erythromycin; in some instances a plasmid which possessed no apparent resistance markers was also transferred. The second strain demonstrated conjugative transfer of penicillin and mupirocin resistance as well as transfer of a plasmid mediating gentamicin resistance, but transfer of erythromycin resistance was not apparently plasmid-mediated.

Chronic urinary tract infection due to Candida utilis.

An elderly male was seen at an outpatient urology clinic over a period of 3 years with repeat urine specimens containing 10(4) to 10(5) CFU of a "Candida species, not C. albicans." The urine specimens were described as infected due to the presence of pyuria, but no antifungal therapy was administered. On two occasions, the patient presented to the emergency room and urine specimens were sent to the clinical microbiology laboratory. On both occasions, a yeast was isolated at concentrations of >10(5) CFU/ml. The organism was identified as the anamorphic yeast Candida utilis (teleomorph: Pichia jadinii) by conventional methods. Molecular methods, including karyotyping and restriction enzyme analysis, confirmed that the isolates were identical and were C. utilis. The patient developed benign prostatic hypertrophy and chronic obstructive pulmonary disease during the 3-year course. This report is the first demonstration of the isolation of the industrially important yeast C. utilis from a urinary tract infection. In the present case, the organism was associated with chronic, symptomatic disease. The significance of this unusual, low-virulence isolate from a case of urinary tract infection is discussed.

Application of molecular typing methods to dermatophyte species that cause skin and nail infections.

Typing methods utilising DNA technology were applied to a collection of Trichophyton mentagrophytes and T. rubrum isolates from skin and nail infections. The methods included restriction enzyme analysis (REA), hybridisation with the DNA probe poly (dG-dT), randomly amplified polymorphic DNA (RAPD) by PCR and restriction analysis of a segment of PCR-amplified rDNA. All these tests successfully differentiated the species, but few intra-species differences were detected. REA demonstrated some isolate variation, but this was limited and difficult to interpret, making it unsuitable as a typing tool. RAPD demonstrated few variations amongst T. mentagrophytes and none in T. rubrum.

Fluoroquinolone resistance in Staphylococcus intermedius.

Four canine isolates of S. intermedius resistant to enrofloxacin were isolated amongst a total of 429 screened. Two of these were shown to exhibit resistance also to marbofloxacin and ciprofloxacin. Whilst molecular studies have shown the mechanism of resistance to these quinolone antibiotics to be similar in a number of staphylococcal species, it was not possible to confirm this mechanism in Staphylococcus intermedius.

Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli.

The mechanism of Pi interaction with phosphate binding protein of Escherichia coli has been investigated using the A197C mutant protein labeled with a coumarin fluorophore (MDCC-PBP), which gives a fluorescence change on binding Pi. A pure preparation of MDCC-PBP was obtained, in which the only significant inhomogeneity is the presence of equal amounts of two diastereoisomers due to the chiral center formed on reaction of the cysteine with the maleimide. These diastereoisomers could not be separated, but Pi binding data suggest that they differ in affinity and fluorescence change. When Pi binds to MDCC-PBP, the fluorescence quantum yield increases 8-fold and the fluorescence intensity at 465 nm increases 13-fold. The kinetics of Pi binding show saturation of the rate at high Pi concentrations, and this together with other information suggests a two-step mechanism with the fluorescence change after binding, concomitant with a conformational change of the protein that closes the cleft containing the Pi binding site. Cleft closure has a rate constant of 317 s-1 (pH 7.0, 5 degrees C), and opening has a rate constant of 4.5 s-1. The fluorescence increase is likely to arise from a change in the hydrophobic environment during this closure as the steady state fluorescence emission (lambdamax and intensity) on Pi binding is mimicked by the addition of ethanol to aqueous solutions of an MDCC-thiol adduct. Fluorescence lifetimes in the absence and presence of Pi were 0.3 and 2.4 ns, respectively, consistent with the change in quantum yield. The rotational correlation time of the coumarin increases only 2-fold from 15 to 26 ns on binding Pi as measured by time-resolved polarization, consistent with the main rotation being determined by the protein even in the open conformation, but with greater local motion. Circular dichroism of the coumarin induced by the protein is weak in the absence of Pi and increases strongly upon saturation by Pi. These data are also consistent with an open to closed conformational model.

Rapid differentiation of closely related Candida species and strains by pyrolysis-mass spectrometry and Fourier transform-infrared spectroscopy.

Two rapid spectroscopic approaches for whole-organism fingerprinting of pyrolysis-mass spectrometry (PyMS) and Fourier transform-infrared spectroscopy (FT-IR) were used to analyze a group of 29 clinical and reference Candida isolates. These strains had been identified by conventional means as belonging to one of the three species Candida albicans, C. dubliniensis (previously reported as atypical C. albicans), and C. stellatoidea (which is also closely related to C. albicans). To observe the relationships of the 29 isolates as judged by PyMS and FT-IR, the spectral data were clustered by discriminant analysis. On visual inspection of the cluster analyses from both methods, three distinct clusters, which were discrete for each of the Candida species, could be seen. Moreover, these phenetic classifications were found to be very similar to those obtained by genotypic studies which examined the HinfI restriction enzyme digestion patterns of genomic DNA and by use of the 27A C. albicans-specific probe. Both spectroscopic techniques are rapid (typically, 2 min for PyMS and 10 s for FT-IR) and were shown to be capable of successfully discriminating between closely related isolates of C. albicans, C. dubliniensis, and C. stellatoidea. We believe that these whole-organism fingerprinting methods could provide opportunities for automation in clinical microbial laboratories, improving turnaround times and the use of resources.

Potential for gene transfer among enterococci from a single patient and the possibility of confounding typing results.

The results of typing methods used in a study of the epidemiology of enterococci in burn patients showed inconsistencies. The possibility that these inconsistencies were the result of gene acquisition or loss was investigated by using 12 isolates of Enterococcus faecalis from a single patient. In vivo and in vitro exchange and loss of genes were observed. The study showed that the typing results for isolates from this patient can be modified by known and demonstrated genetic elements; as a result, the isolates could be divided into between three and seven strains. In the present study, the SmaI digestion patterns gave the most consistent results, correctly identifying the transconjugants as indistinguishable from recipient strain 196R.

Techniques for investigation of an apparent outbreak of infections with Candida glabrata.

A cluster of Candida glabrata isolates recovered from seven patients in an intensive care unit over a 10-week period were compared with a collection of isolates from six epidemiologically distinct outpatients and a reference strain by several DNA typing methods. Restriction enzyme analysis with HinII distinguished 13 strains from the 14 sources and was the method of choice. Pulsed-field gel electrophoresis and random amplification of polymorphic DNA both detected nine types from the 14 sources; however, the results of these two methods did not always correlate. These methods demonstrated that five of the seven patients had distinguishable strains and that cross-infection was unlikely.

Sub-species discrimination, using pyrolysis mass spectrometry and self-organising neural networks, of Propionibacterium acnes isolated from normal human skin.

Curie-point pyrolysis mass spectra were obtained from 30 Propionibacterium acnes strains isolated from the foreheads of six healthy humans. Multivariate analyses and Kohonen artificial neural networks (KANNs), employing unsupervised learning, were used successfully to discriminate between the P.acnes isolates from different individual hosts. The classification of the isolates by KANNs was compared with the more classical multivariate techniques of canonical variates analysis and hierarchical cluster analysis and found to give similar groupings. The combination of pyrolysis mass spectrometry with these numerical methods also showed that more than one strain of P.acnes had been isolated from three of the human hosts.

Application of RAPD and restriction enzyme analysis to the study of oral carriage of Candida albicans.

The genetic similarity of nineteen isolates of Candida albicans from four patients were compared by restriction fragment length polymorphism (RFLP) using EcoRI or HinfI, which both detected five types, and by random amplification of polymorphic DNA (RAPD), which detected three types. Phenotypically unusual isolates also produced distinct patterns with both typing systems demonstrating the carriage of two groups of C. albicans as well as the presence of more than one type in some subjects. Methods of DNA preparation were compared for the production of reproducible patterns; including using the supernatant fluid of boiled intact or spheroplasted cells for RAPD, and DNA precipitated from chloroform extracted cell lysate for RFLP and RAPD. Consistent patterns were produced from the DNA precipitate by RAPD and after an additional precipitation by RFLP, thus removing the necessity for lengthy extraction procedures or the use of toxic chemicals for purification.

Labile antibiotic resistance in Staphylococcus aureus.

Twenty-two isolates of Staphylococcus aureus, recovered from patients over a period of about one year, exhibited low level resistance to mupirocin, developed a salmon pink colour on culture and 21 had the same phage-type. However the plasmid profiles and associated antibiotic resistances differed. Digestion of cellular DNA with SmaI showed that two isolates from a single patient had a markedly different pattern to the remainder, and that six others differed by one band, though these formed groups of one and five isolates. This episode apparently represents a small outbreak of colonization or infection, which would have been missed but for the unusual pigmentation of the isolates recovered, and illustrates the difficulties of relying on a single typing system.

Expression and structural analysis of 14-3-3 proteins.

The 14-3-3 family of proteins plays a role in a wide variety of cellular functions including regulation of protein kinase C and exocytosis. Using antisera specific for the N termini of 14-3-3 isoforms described previously and an additional antiserum specific for the C terminus of epsilon isoform, protease digestion of intact 14-3-3 showed that the N-terminal half of 14-3-3 (a 16 kDa fragment) was an intact, dimeric domain of the protein. Two isoforms of 14-3-3, tau and epsilon, were expressed in E. coli and their secondary structure was shown by circular dichroism to be identical to wild-type protein, and expression of N-terminally-deleted epsilon 14-3-3 protein showed that the N-terminal 26 amino acids are important for dimerization. Intact 14-3-3 is a potent inhibitor of protein kinase C, but the N-terminal domain does not inhibit PKC activity. Site-specific mutagenesis of several regions in the tau isoform of 14-3-3, including the mutation of a putative pseudosubstrate site to a potential substrate sequence, did not alter its inhibitory activity. Intact 14-3-3 proteins are phosphorylated by protein kinase C with a low stoichiometry, but truncated isoforms are phosphorylated much more efficiently by this kinase. This may imply that the proteins may adopt a different structural conformation, possibly upon binding to the membrane, which could modulate their activity. 14-3-3 proteins are found at high concentration on synaptic plasma membranes and this binding is mediated through the N-terminal 12 kDa of 14-3-3.

An in-vitro study of the sterol content and toxin production of Fusarium isolates from mycotic keratitis.

Isolates of Fusarium species from 18 patients with keratomycosis were examined for their C-29 and C-31 sterol content and for their capacity to synthesise mycotoxins. All isolates were resistant to azole antifungal agents in vitro and the sterol contents were indistinguishable. In-vitro toxin production was monitored by gas chromatography-mass spectrometry; 13 isolates produced nivalenol, six produced deoxynivalenol, nine gave T-2 toxin and two showed the presence of diacetoxyscirpenol at different time intervals. However, neither sterol content nor toxin production in vitro appeared to be related to the severity of infections observed in patients.

Identification of mycotoxins in keratomycosis-derived Fusarium isolates by gas chromatography-mass spectrometry.

The production of mycotoxins from Fusarium species has been demonstrated in isolates cultured from patients suffering from keratomycosis. The method employed a combination of thin-layer chromatography directly performed on gel plugs taken from the growth medium, cartridge column chromatography, silylation and gas chromatography on a non-polar stationary phase capillary column linked to mass spectrometry. The sensitivities of detection obtained for a signal-to-noise ratio of 33:1, were 200 pg for single stage GC-MS and 20 pg using tandem GC-MS-MS. Two mycotoxins, diacetoxyscirpenol and T-2 toxin were identified in three cultures.