Molecular epidemiology of Candida albicans strains isolated from the oropharynx of HIV-positive patients at successive clinic visits.

Candida albicans strain diversity and fluconazole resistance were prospectively analyzed in oral strains from 29 adult human immunodeficiency virus (HIV)-positive patients followed for > 1 year who had five or more culture-positive clinic visits. Molecular typing consisted of genomic blots probed with the Ca3 repetitive element. Sixteen patients had one or more episodes of oropharyngeal candidiasis (OPC), 12 (75%) maintained the original genotype, whereas the remaining four patients had a succession of 2-3 genotypes. The original genotype, either alone or mixed with another strain or with non-C. albicans Candida spp., was recovered from oral lesions in 13 of 15 evaluable (86.7%) patients. C. dubliniensis was the infecting yeast in the remaining two patients. Different patterns of fluconazole resistance occurred in three OPC patients. One patient's infecting strain became less susceptible. A second patient was infected with a resistant genotype and a progressively more susceptible minor genotype variant. C. dubliniensis isolates from the third patient varied in susceptibility. Thirteen colonized patients who never developed OPC harbored a greater variety of C. albicans genotypes (2-6) than their infected counterparts (P = 0.35). OPC patients maintained their original endogenous C. albicans strains for prolonged periods, whether or not they demonstrated decreased in vitro susceptibility to fluconazole. The adaptation and maintenance of an endogenous C. albicans strain within its host may be linked to as yet uncharacterized factors.

Candida parapsilosis bloodstream infections in neonatal intensive care unit patients: epidemiologic and laboratory confirmation of a common source outbreak.

BACKGROUND: Candida parapsilosis is a common cause of sporadic and epidemic infections in neonatal intensive care units (NICUs). When a cluster of C. parapsilosis bloodstream infections occurred in NICU patients in a hospital in Louisiana, it provided us with the opportunity to conduct an epidemiologic investigation and to apply newly developed molecular typing techniques. METHODS: A case-patient was defined as any NICU patient at Louisiana State University Medical Center, University Hospital, with a blood culture positive for C. parapsilosis during July 20 to 27, 1991. To identify risk factors for C. parapsilosis bloodstream infection, a cohort study of all NICU infants admitted during July 17 to 27, 1991, was performed. Electrophoretic karyotyping was used to assess the relatedness of C. parapsilosis isolates. RESULTS: The receipt of liquid glycerin given as a suppository was identified as a risk factor (relative risk, 31.2; 95% confidence intervals, 4.3 to 226.8). Glycerin was supplied to the NICU in a 16-oz multidose bottle. Bottles used at the time of the outbreak were not available for culture. All six available isolates from four case-patients had identical chromosomal banding patterns; six University Hospital non-outbreak isolates had different banding patterns. CONCLUSIONS: This study demonstrates the utility of combined epidemiologic and laboratory techniques in identifying a novel common source for a C. parapsilosis bloodstream infection outbreak and illustrates that extreme caution should be exercised when using multidose medications in more than one patient.

Comparison of multilocus enzyme electrophoresis and random amplified polymorphic DNA analysis for molecular subtyping of Cryptococcus neoformans. The Cryplococcal Disease Active Surveillance Group.

We evaluated multilocus enzyme electrophoresis (MEE) and random amplified polymorphic DNA (RAPD) for their usefulness in subtyping 344 Cryptococcus neoformans clinical isolates obtained from four U.S. metropolitan areas in 1992 to 1994. MEE and RAPD with five primers both discriminated between the two varieties of C. neofromans. MEE divided C. neoformans var. neoformans isolates into 15 enzyme electrophoretic subtypes (ETs) arranged in three complexes. The predominant ET 1 complex contained 10 ETs, with isolates from 70% of patients in 1 ET. RAPD with five primers further sorted this predominant ET into 19 subtypes, with 60% of isolates sorting into three RAPD types. The ET 8 MEE complex, containing three ETs, could not be divided further by RAPD. The ET 7 complex (two ETs) included isolates from all serotype AD patients. Although both MEE and RAPD identified isolates of C. neoformans var. gattii, neither distinguished between serotypes B and C. These results showed that the two C. neoformans varieties could be identified by MEE or RAPD profile as well as by biochemical methods. RAPD improved the discriminatory power of MEE for isolates within the ET 1 complex but with other ETs offered little additional sensitivity over MEE and was less sensitive than MEE with isolates of C. neoformans var. gattii. This information will be useful in identifying particular environmental sources of disease-causing exposures, in seeking clusters of cases, and in determining whether an infecting strain changes over time.

Nucleotide sequence analysis of the 5.8S rDNA and adjacent ITS2 region of Candida albicans and related species.

We have determined the nucleotide sequence for the DNA encoding the 5.8S RNAs and downstream internal transcribed spacer (ITS2) regions for Candida albicans and the taxonomically related species C. parapsilosis, C. tropicalis, C. glabrata and C. krusei. Phylogenetic analysis of all known fungal 5.8S RNA sequences revealed a close relationship between C. tropicalis and C. parapsilosis, and to a lesser extent C. albicans within the yeast-like fungi. This group can itself be delineated from predominantly filamentous species. The more distal relationships between Candida (Torulopsis) glabrata and C. krusei support previous findings based on small (18S) ribosomal RNA sequence analysis, suggesting a greater degree of evolutionary divergence of these species from the C. albicans group. Among strains of C. albicans we observed conservation of the ITS2 region at the nucleotide level. Conservation was also observed for a more limited number of C. parapsilosis strains. Although the 3' region of the ITS spacer was species specific, sequence homology was observed in the 5' end within the albicans/parapsilosis/tropicalis group. Our findings suggest a rapid approach to species identification through the use of non-conserved regions flanked by highly conserved, functional domains.

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.

Genomic heterogeneity in the yeast Candida parapsilosis.

Candida parapsilosis shows a wide intraspecies variation in chromosome/homolog size distribution. As a prerequisite for delineating modes of transmission, we have undertaken an analysis of genetic variation at different levels. In the present study we have observed that a majority of isolates display similar electrophoretic karyotype patterns consistent for the species, with variations in the smaller group of chromosomes. In two strains we observed phenotypic "switching"; one of these also exhibited a mixed karyotypic subpopulation. In contrast, a few isolates displayed a greater degree of chromosome/homolog size variation. We also observed, through randomly amplified polymorphic DNA (RAPD) analysis, results consistent with those of pulsed-field electrophoresis. Isolates displaying a high degree of chromosome/homolog variation also displayed a high degree of variation in genomic "fingerprints". Polymorphisms, although present, were much reduced in the majority of isolates. These parallel observations suggest a common underlying mechanism. Our results are consistent with the hypothesis that chromosome-sized variations in C. parapsilosis are due to random genetic events. A similar mechanism has been hypothesized for the taxonomically related yeast Candida albicans.

Monoclonal antibodies against Candida tropicalis mannan: antigen detection by enzyme immunoassay and immunofluorescence.

Three strains of mice were immunized with Candida tropicalis cell walls, and antibodies against mannan were detected by indirect enzyme immunoassay (EIA) in 3 of 9 BALB/c mice, 4 of 11 C57BL/6 mice, and 4 of 8 CFW mice. Responding mice produced immunoglobulin M (IgM), but IgG was not detected in their sera. Fusion of the high-responder BALB/c mouse with a plasmacytoma cell line resulted in 41 clones secreting antimannan monoclonal antibodies (MAbs). Four clones selected for propagation included one IgM and one IgG MAb that reacted with mannans of Candida albicans serotypes A and B and of C. tropicalis and two IgM MAbs specific for an epitope only in the mannans of C. albicans serotype A and C. tropicalis. One of the IgM MAbs, CB6, was an effective substitute for rabbit antibodies in the double-antibody sandwich EIA to detect antigenemia produced in rabbits infected with C. albicans A or C. tropicalis. It could function either as the peroxidase-conjugated indicator antibody or as the capture antibody. Two MAbs, CB6 (C. tropicalis and C. albicans A specific) and AC3 (C. tropicalis and C. albicans A and B specific), functioned in place of polyclonal antisera in the serotyping of C. albicans by immunofluorescence. There was 95.8% agreement in the results of serotyping using MAbs as reagents compared with rabbit antisera. Competitive inhibition in EIA between CB6 and monospecific antisera against C. albicans factors 1, 4, and 6 indicated that CB6 binds to an epitope which is probably factor 6. Serologic similarity between factor 4 and the binding site of MAb AC3 was also determined.

Antigenemia in rabbits infected with Candida albicans serotype B: detection by enzyme immunoassay and preliminary characterization of the antigen.

Peroxidase-labeled IgG against Candida albicans serotype A cell walls failed to detect antigenemia in rabbits infected with C. albicans type B. Therefore type B antisera were produced in rabbits immunized with either type B cell walls; whole blastoconidia heated and killed at 60 degrees C (HK); a sublethal intravenous dosage of 10(6) colony forming units (c.f.u.); or a normally lethal dose of 10(7) c.f.u. injected into subcutaneously implanted plastic chambers. The four antisera were conjugated to peroxidase for a double antibody sandwich enzyme immunoassay. Sixteen rabbits, immunosuppressed with cortisone, received 10(7) type B blastoconidia intravenously. On day 4 their kidneys contained between 3.4 X 10(8) and 1.4 X 10(11) c.f.u. Antigenemia was detected only with the conjugated anti-HK-IgG. A mean concentration of 246 ng ml-1 of circulating antigen was detected only when serum-antigen complexes were dissociated by boiling in EDTA. The properties of the circulating antigen were as follows: relative molecular weight between 50 000 and 100 000 da; stable to boiling and proteinases, labile to sodium periodate oxidation, and bound by concanavalin A. These properties are consistent with a polysaccharide or glycoprotein, probably the mannoprotein of the cell wall. Antigenic specificity was probed further using IgG produced against heat-killed C. albicans serotype A. Antigenemia resulting from infection with serotype B was detected in 5 of 5 rabbits tested with the anti-HK-serotype B IgG but only with 3 of 5 tested with the anti-HK-serotype A IgG. In addition, the concentrations detected with serotype B reagents were 31 times higher.

Comparison of enzyme immunoassay and gas-liquid chromatography for the rapid diagnosis of invasive candidiasis in cancer patients.

Three proposed quantitative markers for candidiasis, arabinitol, mannose, and mannan in serum, are compared in 50 normal blood donors and 38 high-risk patients, 23 with and 15 without invasive candidiasis. Arabinitol concentrations in serum, the arabinitol/creatinine ratio, and mannose concentrations in serum were significantly greater in the 15 patients without candidiasis than in the normal blood donors (P less than 0.05). The sensitivities and specificities were 26 and 87% for arabinitol, 13 and 93% for the arabinitol/creatinine ratio, and 39 and 87% for mannose. On the other hand, mannan concentrations in serum were less than 1 ng/ml in normal blood donors and patients without candidiasis (P = 0.344), and the sensitivity and specificity were 65 and 100%, respectively. Of 23 patients with proven or probable candidiasis, 16 had mannan levels in serum greater than the mean + 2 standard deviations (0.46 ng/ml) for the 15 controls. In 16 patients with invasive candidiasis and positive blood cultures for the Candida spp., only 13 had elevated levels of at least one of the three markers. The arabinitol/creatinine ratio, the mannose level, and the mannan level became elevated an average of 4 days before, 1 day before, and on the same day that the blood cultures were drawn, respectively. Conversely, mannan was detected in the sera of six of seven patients with invasive candidiasis and negative blood cultures. We conclude that the best approach to diagnosing invasive candidiasis involves obtaining blood cultures and carrying out serial assays for mannan in serum.

Comparison of serum mannan, arabinitol, and mannose in experimental disseminated candidiasis.

Concentrations of arabinitol, mannose, and mannan in serum have independently been reported to be elevated in patients with invasive candidiasis. These three marker substances were compared in a rabbit model. Twelve rabbits, immunosuppressed with cortisone, were infected intravenously with Candida albicans 3181A. Six uninfected control animals also received cortisone, and four rabbits were neither infected nor immunosuppressed. Blood samples, drawn from 2 days before to 14 days after infection, were assayed for serum mannan by sandwich enzyme immunoassay, antibodies to mannan by indirect enzyme immunoassay, arabinitol and mannose by gas-liquid chromatography, and serum creatinine. Serum mannan, negative before infection, peaked in all infected animals 4 days after infection (mean, 18 ng/ml) and decreased thereafter. Significant increases (2 standard deviations greater than mean in normals) in arabinitol, the arabinitol/creatinine ratio, and mannose were found in 12, 8, and 12 of the infected rabbits, respectively, but also in all 6 uninfected animals receiving cortisone. Only serum mannan was specific in this immunosuppressed rabbit model.

Simultaneous determination of arabinitol and mannose by gas-liquid chromatography in experimental candidiasis.

A method is described for the simultaneous quantitation of D-arabinitol and D-mannose in serum by gas-liquid chromatography as an aid for the diagnosis of disseminated candidiasis. Both variables were observed as per-O-acetylated aldononitrile derivatives in each chromatographic run of sera from immunosuppressed rabbits experimentally infected with Candida albicans 3181A.

Dissociation of mannan--serum complexes and detection of Candida albicans mannan by enzyme immunoassay variations.

Candida albicans mannan was added to normal human sera and the resulting complexes were dissociated by boiling (boil) with EDTA or by alkali treatment (bead method). The mannan released was detected by "sandwich" enzyme immunoassay (EIA) or by EIA inhibition. Each EIA took 2.3 h to perform. The total time for the boil-EIA combination was 2.7 h and for the bead-EIA, 3.8 h. The temperature favorable for antigen--antibody incubation was 4 degrees C. The sandwich EIAs were preferable to EIA inhibition because absorbance was directly proportional to mannan concentration, within-run variation was decreased, and accuracy was increased. The boil-sandwich EIA had the highest sensitivity in the 12.5 to 200 micrograms/L range.