Interlaboratory reproducibility of a single-locus sequence-based method for strain typing of Aspergillus fumigatus.

Seven international laboratories tested the recently proposed single-locus typing strategy for Aspergillus fumigatus subtyping for interlaboratory reproducibility. Comparative sequence analyses of portions of the locus AFUA_3G08990, encoding a putative cell surface protein (denoted CSP), was performed with a panel of Aspergillus isolates. Each laboratory followed very different protocols for extraction of DNA, PCR, and sequencing. Results revealed that the CSP typing method was a reproducible and portable strain typing method.

Molecular methods for the identification of Aspergillus species.

Invasive aspergillosis (IA) is a leading cause of morbidity and mortality in immunocompromised hosts. In some institutions, species of Aspergillus less susceptible to amphotericin B than Aspergillus fumigatus are becoming more common, making an accurate identification of species important. However, species identification has traditionally relied on macroscopic colony characteristics and microscopic morphology, which may require several days of culture. Additional sub-culturing on specialized media may be required to induce conidia formation; in some cases conidia may never form, confounding identification. Therefore, rapid, nucleic acid-based methods that identify species of Aspergillus independent of morphology are now being developed to augment or replace phenotypic identification methods. The most successful methods to date have employed polymerase chain reaction (PCR) amplification of target sequences within the ribosomal RNA gene complex, including the 28S ribosomal subunit (D1-D2 region) and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). We therefore developed a PCR-based assay to differentiate medically important species of Aspergillus from one another, and from other opportunistic moulds and yeasts, by employing universal, pan-fungal primers directed to conserved ribosomal genes and species-specific DNA probes directed to the highly variable ITS2 region. Amplicons were then detected in a simple, colorimetric enzyme immunoassay format (PCR-EIA). DNA sequencing of the ITS1 and ITS2 regions and of the D1-D2 region was also conducted for the differentiation of species by comparative GenBank sequence analysis. The PCR-EIA method was found to be rapid, sensitive, and specific for the identification and differentiation of the most medically important species of Aspergillus. In addition, methods to identify species of Aspergillus by comparative GenBank sequence analysis were found to be more reliable using the ITS1 and ITS2 regions than the D1-D2 region.

Rapid and unequivocal differentiation of Candida dubliniensis from other Candida species using species-specific DNA probes: comparison with phenotypic identification methods.

Candida dubliniensis is a recently described opportunistic pathogen which shares many phenotypic characteristics with Candida albicans but which has been reported to rapidly acquire resistance to azole antifungal drugs. Therefore, differentiation of C. dubliniensis from C. albicans becomes important to better understand the clinical significance and epidemiologic role of C. dubliniensis in candidiasis. We compared phenotypic methods for the differentiation of C. dubliniensis from C. albicans (i.e. the ability to grow at elevated temperatures, colony color on CHROMagar Candida medium, and carbohydrate assimilation patterns) to amplify the results of a polymerase chain reaction (PCR) assay using universal fungal primers to the internal transcribed spacer 2 (ITS2) region of rDNA and species-specific DNA probes in an enzyme immunoassay format (PCR-EIA). DNA sequencing of the ITS1 rDNA region was also conducted. The C. dubliniensis ITS2 probe correctly identified all C. dubliniensis isolates without cross-reaction with any other Candida species tested (mean A(650 nm) +/- SE, C. dubliniensis probe with C. dubliniensis DNA, 0.372 +/- 0.01, n = 22; C. dubliniensis probe with other Candida species DNA, 0.001 +/- 0.02 n = 16, P < 0.001). All other Candida species tested (C. albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis) were also correctly identified by the PCR-EIA without any detectable cross-reactions among species. Phenotypically, C. dubliniensis isolates demonstrated an increased sensitivity to heat compared to C. albicans isolates. At 42 degrees C, only 50% of C. dubliniensis isolates grew compared to 73% of C. albicans isolates and, at 45 degrees C, 91% of C. dubliniensis isolates failed to grow compared to 64% of C. albicans isolates. C. albicans was more likely to demonstrate a dark green or blue green colony color on CHROMagar Candida medium obtained from Becton Dickinson (i.e. 100% of C. albicans isolates were dark green or blue green versus 64% of C. dubliniensis isolates) whereas no difference in the percentage of C. albicans or C. dubliniensis isolates producing dark green or blue green colony color was detected using CHROMagar Candida medium from Hardy Diagnostics (82% for both species). The API 20C AUX carbohydrate assimilation system incorrectly identified C. dubliniensis as C. albicans in all but three cases: remaining isolates were misidentified as C. albicans/C. tropicalis, C. tropicalis/C. albicans, and Candida lusitaniae/C. albicans. In all, 82% of C. albicans isolates and 100% of C. dubliniensis isolates assimilated trehalose; the latter finding was opposite to that reported for C. dubliniensis in the API 20C AUX profile index. Xylose and alpha-methyl-D-glucoside assimilation, respectively, were negative for 100 and 95% of C. dubliniensis isolates and positive for 100 and 91% of C. albicans isolates, confirming earlier reports that assimilation results for xylose and alpha-methyl-D-glucoside may be helpful in the discrimination of these two species. However, conventional phenotypic species identification tests required days for completion, whereas the PCR-EIA could be completed in a matter of hours. In addition, identification of Candida species by ITS1 rDNA sequencing gave 100% correspondence to the results obtained by the PCR-EIA, confirming the specificity of the PCR-EIA method. These data indicate that although a combination of phenotypic methods may help differentiate C. dubliniensis from C. albicans to some extent, the PCR-EIA can provide a simple, rapid, and unequivocal identification of the most medically important Candida species in a single test.

Rapid identification of dimorphic and yeast-like fungal pathogens using specific DNA probes.

Specific oligonucleotide probes were developed to identify medically important fungi that display yeast-like morphology in vivo. Universal fungal primers ITS1 and ITS4, directed to the conserved regions of ribosomal DNA, were used to amplify DNA from Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, Penicillium marneffei, Sporothrix schenckii, Cryptococcus neoformans, five Candida species, and Pneumocystis carinii. Specific oligonucleotide probes to identify these fungi, as well as a probe to detect all dimorphic, systemic pathogens, were developed. PCR amplicons were detected colorimetrically in an enzyme immunoassay format. The dimorphic probe hybridized with DNA from H. capsulatum, B. dermatitidis, C. immitis, P. brasiliensis, and P. marneffei but not with DNA from nondimorphic fungi. Specific probes for H. capsulatum, B. dermatitidis, C. immitis, P. brasiliensis, P. marneffei, S. schenckii, C. neoformans, and P. carinii hybridized with homologous but not heterologous DNA. Minor cross-reactivity was observed for the B. dermititidis probe used against C. immitis DNA and for the H. capsulatum probe used against Candida albicans DNA. However, the C. immitis probe did not cross-react with B. dermititidis DNA, nor did the dimorphic probe hybridize with C. albicans DNA. Therefore, these fungi could be differentiated by a process of elimination. In conclusion, probes developed to yeast-like pathogens were found to be highly specific and should prove to be useful in differentiating these organisms in the clinical setting.

Comparison of commercial latex agglutination and sandwich enzyme immunoassays with a competitive binding inhibition enzyme immunoassay for detection of antigenemia and antigenuria in a rabbit model of invasive aspergillosis.

A commercial latex agglutination assay (LA) and a sandwich enzyme immunoassay (SEIA) (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) were compared with a competitive binding inhibition assay (enzyme immunoassay [EIA]) to determine the potential uses and limitations of these antigen detection tests for the sensitive, specific, and rapid diagnosis of invasive aspergillosis (IA). Toward this end, well-characterized serum and urine specimens were obtained by using a rabbit model of IA. Serially collected serum or urine specimens were obtained daily from control rabbits or from rabbits immunosuppressed and infected systemically with Aspergillus fumigatus. By 4 days after infection, EIA, LA, and SEIA detected antigen in the sera of 93, 93, and 100% of A. fumigatus-infected rabbits, respectively, whereas antigen was detected in the urine of 93, 100, and 100% of the rabbits, respectively. False-positive results for non-A. fumigatus-infected rabbits for EIA, LA, and SEIA were as follows: for serum, 14, 11, and 23%, respectively; for urine, 14, 84, and 90%, respectively. Therefore, although the sensitivities of all three tests were similar, the specificity was generally greater for EIA than for LA or SEIA. Infection was also detected earlier by EIA, by which the serum of 53% of A. fumigatus-infected rabbits was positive as early as 1 day after infection, whereas the serum of only 27% of the rabbits tested by LA was positive. Although the serum of 92% of A. fumigatus-infected rabbits was positive by SEIA as early as 1 day after infection, the serum of a high percentage (50%) was false positive before infection. The urine of 21% of A. fumigatus-infected rabbits was positive by EIA as early as 1 day after infection, and the urine of none of the rabbits was false positive before infection. When EIA results for urine specimens were combined with those for serum, sensitivity was improved (i.e., 67% of rabbits were positive by 1 day after infection and only one rabbit gave a false-positive result). A total of 93% of A. fumigatus-infected rabbits were positive for antigen in urine as early as 1 day after infection and the urine of 100% of the rabbits was positive by SEIA. However, before infection, 79% of A. fumigatus-infected rabbits were false positive for antigen in urine by LA and 90% were false positive for antigen in urine by SEIA. These data indicate that the EIA has the potential to be used to diagnose IA with both serum and urine specimens and to detect a greater number of infections earlier with greater specificity than the specificities achieved with the commercial tests.

Enhanced extracellular production of aspartyl proteinase, a virulence factor, by Candida albicans isolates following growth in subinhibitory concentrations of fluconazole.

We examined the production of secreted aspartyl proteinase (Sap), a putative virulence factor of Candida albicans, by a series of 17 isolates representing a single strain obtained from the oral cavity of an AIDS patient before and after the development of clinical and in vitro resistance to fluconazole. Isolates were grown in Sap-inducing yeast carbon base-bovine serum albumin medium containing 0, 0.25, 0.5, or 1 MIC of fluconazole, and cultures were sampled daily for 14 days to determine extracellular Sap activity by enzymatic degradation of bovine serum albumin. Extracellular Sap activity was significantly decreased in a dose-dependent manner for the most fluconazole-susceptible isolate (MIC, 1.0 microg/ml) and significantly increased in a dose-dependent manner for the most fluconazole-resistant isolate (MIC, >64 microg/ml). Enhanced extracellular Sap production could not be attributed to cell death or nonspecific release of Sap, because there was no reduction in the number of CFU and no significant release of enolase, a constitutive enzyme of the glycolytic pathway. Conversely, intracellular Sap concentrations were significantly increased in a dose-dependent manner in the most fluconazole-susceptible isolate and decreased in the most fluconazole-resistant isolate. Enhanced Sap production correlated with the overexpression of a gene encoding a multidrug resistance (MDR1) efflux pump occurring in these isolates. These data indicate that exposure to subinhibitory concentrations of fluconazole can result in enhanced extracellular production of Sap by isolates with the capacity to overexpress MDR1 and imply that patients infected with these isolates and subsequently treated with suboptimal doses of fluconazole may experience enhanced C. albicans virulence in vivo.

Purification and characterization of the extracellular aspartyl proteinase of Candida albicans: removal of extraneous proteins and cell wall mannoprotein and evidence for lack of glycosylation.

Aspartyl proteinase (AP) is an extracellular enzyme of Candida albicans implicated as a pathogenic factor. Previous reports on the purification and characterization of AP suggested that a single DEAE-Sephadex chromatographic step was sufficient for the removal of extraneous proteins and that the final product was glycosylated. We purified AP using a chromatographic series consisting of DEAE-Sephadex A25, Sephadex G75 and rechromatography on DEAE-Sephadex A25. Use of DEAE-Sephadex alone did not remove extraneous proteins and removed little contaminating mannoprotein (MP). The addition of a Sephadex G75 column to the purification scheme removed the majority of contaminating MP and proteins. The final DEAE-Sephadex A25 chromatographic step resulted in (a) removal of detectable extraneous proteins, (b) removal of immunologically detectable MP by dot blot and Western blot enzyme immunoassay, (c) loss of periodic acid-silver stain positivity, and (d) a high AP yield (1295 U l-1) and specific activity (1749 U mg-1). We conclude that a single DEAE-Sephadex A25 purification step is insufficient to remove extraneous proteins and MP, which could interfere with the production of AP-specific antibodies and the dissection of moieties responsible for immune reactivity. Reports of periodic acid-Schiff or anthrone positivity of AP preparations may reflect the presence of extraneous MP, which can be removed by the chromatographic series we describe.

Heterogeneity of the purified extracellular aspartyl proteinase from Candida albicans: characterization with monoclonal antibodies and N-terminal amino acid sequence analysis.

Three dominant proteins (41, 48, and 49 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in purified preparations of the extracellular aspartyl proteinase (AP) of Candida albicans. All three proteins bound to the specific carboxyl proteinase ligand, pepstatin A, and were associated with maximum AP activity. The N-terminal amino acid sequence for the 48- and 49-kDa proteins matched that reported by others for AP, whereas the sequence for the 41-kDa protein was unique and was not homologous to any known protein. Time course studies demonstrated the simultaneous presence of all three proteins, supporting evidence that the 41- and 48-kDa proteins were not breakdown products of AP. Previous studies did not detect carbohydrate in SDS-polyacrylamide gels of purified AP preparations stained with periodic acid and silver, making glycosylation an unlikely explanation for the observed differences in the molecular masses of the proteins. Some monoclonal antibodies directed against the 49-kDa protein reacted with the 41- and 48-kDa proteins, indicating cross-reactive epitopes. Other monoclonal antibodies, however, reacted only with the 49-kDa protein. We conclude that three pepstatin A-binding proteins occur in purified AP preparations: two have the same amino acid N terminus as that reported for AP, whereas the third has a unique sequence. All three proteins should be considered when undertaking studies to determine the role of AP in candidal pathogenesis or when preparing specific antibodies for antigen capture assays.

Evaluation of cation exchange chromatography for the isolation of M glycoprotein from histoplasmin.

Cation exchange chromatography was evaluated to purify the M antigen from histoplasmin (HMIN). Two H and M antigen-containing fractions, soluble (S) and precipitate (PP), resulted from the initial 0.025 M, pH 3.5 citrate buffer dialysis step. The PP fraction contained 62% of the M antigen activity and was resolubilized. Both fractions were chromatographed on CM Sepharose CL-6B. Polysaccharide C antigen was abundant in the S fraction and most of it did not bind to CM Sepharose. M antigen-enriched fractions were eluted with 0.5 M NaCl. Re-chromatography of the relevant S fraction (S-II) and PP fraction (PP-II) by linear gradient fast protein liquid chromatography (FPLC) removed protein and C impurities. M antigen purified by FPLC from the PP-II fraction was depleted of other antigens when Western blots were probed with anti-M, anti-H and anti-C monoclonal antibodies (Mabs). M antigen was identified as a 94 kDa glycoprotein containing a specific-protein epitope and an epitope that reacted with a Mab against the polysaccharide C antigen. M antigen can be purified from HMIN by tandem cation exchange chromatography of the precipitable fraction on an open CM Sepharose CL-6B column followed by linear gradient FPLC.

Western immunoblot analysis and serologic characterization of Blastomyces dermatitidis yeast form extracellular antigens.

A major 98-kDa extracellular protein antigen of Blastomyces dermatitidis was shown in Western blot (immunoblot) analysis to be highly reactive with serum antibodies from patients with blastomycosis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of yeast form B. dermatitidis culture filtrates and cell extracts demonstrated over 50 proteins, only 24 of which were immunoreactive. Of these, a 98-kDa protein was found to be the most specific and was isolated. This protein was found in both broth culture filtrates and extracts of yeast forms. Western blot tests with this antigen detected serum antibodies in 91% of 32 patients with clinically proven blastomycosis, in contrast to an enzyme-linked immunosorbent assay (ELISA) with DEAE-purified antigen, which detected 88% of the cases. The Western blot test also exhibited lower reactivity with a panel of sera from patients with heterologous fungal infections that were cross-reactive in the ELISA with DEAE-purified B. dermatitidis antigen. The 98-kDa protein electroeluted from polyacrylamide gels was identical to the diagnostic A antigen used in the blastomycosis immunodiffusion test. Comparison of the 98-kDa antigen with a previously described 120-kDa yeast form cell wall protein antigen of B. dermatitidis showed that these two antigens are almost identical.

Role of the Cell Surface of Methanosarcina mazei in Cell Aggregation.

Colonial aggregates of Methanosarcina (= Methanococcus) mazei were examined with scanning and transmission electron microscopy. Cells are irregular and grouped into multicellular sarcinal colonies, which may disaggregate in older cultures. The protoplast is bounded by a typical trilaminar plasma membrane, outside of which is a matrix of loose fibrils. The presence and compactness of matrix material are responsible for the close packing of cells, and colony disaggregation seems to be the result of matrix shedding and degradation. The cell envelope contains complex hetero polysaccharides of N-acetylgalactosamine and galacturonic and glucuronic acids. Polymers extruded by M. mazei are likely quite adhesive in nature, accounting for its strong adherence to surfaces and hardiness compared with many other methanogens.

Chemical analyses of membranes isolated from Streptococcus mutans BHT.

Cell membranes of Streptococcus mutans BHT (serotype b) were prepared following glass-bead disruption or mutanolysin digestion of whole cells. Major constituents of purified BHT membranes included: protein (60-65%), fatty acids (10%), glucose (3%), and phosphorus (0.5%). The principal amino acids measured were glutamate, aspartate, lysine, alanine, and leucine. The principal fatty acids measured were octadecenoic, palmitoleic, palmitic, and eicosenoic acids; smaller amounts of eicosanoic acid were also detected. Chemical analyses of membranes from cells grown to four different growth phases revealed no major shifts in composition during batch growth under our experimental conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these four membrane preparations confirmed the apparent compositional stability of cell membranes during growth.

Mutanolysin-induced spheroplasts of Streptococcus mutants are true protoplasts.

A method is described for the preparation of protoplasts of Streptococcus mutans BHT. The muralytic enzyme mutanolysin was prepared free of contaminating proteinases and shown to completely dissolve cell walls of this strain. Whole cells were converted to stabilizable protoplasts by using the enzyme in an isotonic medium containing 40% raffinose. Experiments using [3H]thymidine and [14C]leucine as cytoplasmic pool markers revealed only minimal (10%) leakage during a 1-h incubation. Examination by electron microscopy revealed the apparent absence of structural cell wall on the enlarged spherical bodies. Quantitative chemical analyses of membranes prepared by lysing protoplasts demonstrated only very small amounts of rhamnose and trace amounts of galactose. These sugars are the principal components of the BHT cell wall polysaccharide. Also, there were only small amounts of peptidoglycan components (e.g., N-acetylglucosamine) in the purified membranes obtained by this method.

Phenotypic stability of the cell wall of Streptococcus mutans Ingbritt grown under various conditions.

Quantitative analyses of cell walls from Streptococcus mutans Ingbritt grown under carbohydrate limitation in the chemostat showed that growth conditions had no statistically significant effect on the composition of polysaccharide, peptidoglycan, or the proportion of polysaccharide in the cell wall. Lysis of cell wall preparations with a muramidase supported this conclusion and further indicated that there was little difference in their overall structure. In contrast, there was a consistent difference between the rates of lysis by this enzyme of organisms grown in 0.2% glucose and 0.5% glucose. Extremes of pH or dilution rate essentially did not influence the immunogenicity of type c antigen in whole organisms irrespective of whether the carbohydrate source was glucose or sucrose. However, differences were found in the immunogenicity of lipoteichoic acid under similar circumstances. The results indicated there was an inherent phenotypic stability in the cell walls of S. mutans Ingbritt despite changes in pH, generation time, and carbohydrate source, and that any changes that did occur were probably due to associated cell-surface components.