Multiplex PCR-ligation detection reaction assay for simultaneous detection of drug resistance and toxin genes from Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium.

A multiplex PCR-ligation detection reaction (PCR-LDR) assay was developed for rapid detection of methicillin, tetracycline, and vancomycin resistance, as well as toxic shock toxin and Panton-Valentine leukocidin. The assay was tested on 470 positive blood culture bottles containing Staphylococcus aureus or enterococci. PCR-LDR exhibited a sensitivity and specificity of > or = 98% for all components except tetracycline resistance, which had a sensitivity of 94.7%. Rapid and sensitive detection of antimicrobial resistance and virulence genes could help guide therapy and appropriate infection control measures.

Rapid detection of Clostridium difficile in stool using the VIDASR C. difficile Toxin A II assay.

A rapid laboratory diagnosis of Clostridium difficile-associated diarrhea (CDAD) is important in patient management and in the administration of appropriate therapeutic modalities. The VIDAS(R) C. difficile Toxin A II (CDA 2) assay (bioMerieux, Inc., Hazelwood, MO) was compared with the cell culture cytotoxicity assay (CCA) for the rapid detection of C. difficile in stool from patients in whom C. difficile infection was suspected. Thirty-eight consecutively collected CCA-positive stool specimens, and 33 CCA-negative stool specimens were tested by the CDA 2 assay. Where appropriate, discordant specimens were repeated and/or tested by isolation utilizing cycloserine-cefoxitin-fructose agar (CCFA). Among 12 discordant stool specimens, 7 were VIDAS(R)-/cytotoxicity+, 2 were VIDAS(R) equivocal (E)/cytotoxicity+, 2 were VIDAS(R) E/cytotoxicity-, and 1 was VIDAS(R)+/cytotoxicity-. One VIDAS(R) E/cytotoxicity+ lacked sufficient stool to be repeated. From the single VIDAS(R)+/cytotoxicity- specimen, C. sordelli was isolated. Specimens that were equivocal by VIDAS(R), were omitted from incorporation into this study's test parameters. The sensitivity, specificity, positive and negative predictive values for the CDA 2 assay were 80.6, 96.8, 96.7, and 81.1%, respectively. The specimens which yielded false negative VIDAS(R) results had low levels of toxin based on endpoint titrations using the cytotoxicity assay. Although the CDA 2 assay displayed a reduced sensitivity compared with the CCA, the automated assay is rapid (results promulgated within 2 h), with computer generated readings obviating visual interpretations. Recognition of the CDA 2 assay's limitations is important to addressing this test's clinical utility.

Isolation of Cryptococcus neoformans chromosome-specific probes using expressed sequence tags.

Clinical and environmental isolates of Cryptococcus neoformans exhibit a high degree of karyotypic variability. Analysis of the molecular basis of karyotypic differences requires a large set of chromosome-specific probes. We have determined the chromosomal distribution of a set of randomly selected C. neoformans cDNA clones and have explored the feasibility of identifying these clones through partial DNA sequencing. Forty-four randomly selected cDNA clones were labelled and hybridized to PFGE blots of C. neoformans. Expressed sequence tags were generated by sequencing the 5'-end of each clone. Thirty-five clones hybridized to single bands on PFGE blots. At least seven chromosomes were recognized by these probes. Homology searches identified potential homologs of several groups of proteins not previously studied in C. neoformans. PFGE hybridization and sequencing of random cDNA clones is an efficient method for identifying chromosomal-specific probes in fungi that lack extensive sets of genetic markers.

Molecular subtypes and antifungal susceptibilities of serial Cryptococcus neoformans isolates in human immunodeficiency virus-associated Cryptococcosis. Cryptococcal Disease Active Surveillance Group.

Serial isolates of Cryptococcus neoformans from 33 human immunodeficiency virus-infected patients with cryptococcosis were analyzed to determine whether persistence might result from reinfection with a new cryptococcal strain or acquisition of antifungal resistance. Isolates were subtyped by multilocus enzyme electrophoresis (MEE), electrophoretic karyotyping (EK), random-amplified polymorphic DNA (RAPD), and the CNRE-1 DNA probe, MICs of amphotericin B, fluconazole, and 5-fluorocytosine were determined. No changes in MEE or RAPD subtypes were detected in serial isolates from any patient. Isolates from 8 patients (24%) showed alterations in EK only (mobility change in two or more bands) but not with any other subtyping method. MICs did not change significantly in isolates from 30 patients. In 1 case, the fluconazole MIC increased stepwise over 18 months, suggesting development of resistance. These overall invariant subtyping and MIC results confirm previous studies suggesting that persistent cryptococcal infection is due to relapse rather than reinfection or antifungal drug resistance.

Catheter-associated fungemia due to Wangiella (Exophiala) dermatitidis.

We describe a case of catheter-associated Wangiella (Exophiala) dermatitidis fungemia in a human immunodeficiency virus-infected child who was successfully treated with antifungal therapy and catheter removal. Catheter-associated W. dermatitidis fungemia appears to be distinct from previously described cases of disseminated infection with organ involvement.

Genetic relatedness of Cryptococcus neoformans clinical isolates grouped with the repetitive DNA probe CNRE-1.

Cryptococcus neoformans isolates from eight patients with cryptococcal infection were previously assigned into three groups on the basis of repetitive DNA probe (CNRE-1) restriction fragment length polymorphisms. These groups accounted for a disproportionate number of recent clinical isolates in New York City. To further examine the genetic relatedness of isolates within and across CNRE-1 groups, the DNA sequence of the 779-base URA5 gene from each strain was amplified and sequenced. The number of nucleotide differences occurred in the third codon position or in introns. Pairwise comparisons revealed average nucleotide differences within a CNRE-1 group of 4.8 +/- 2.6 (n = 8) and between CNRE-1 groups of 21.9 +/- 7.0 (n =20) (P <0.001) Analysis of URA5 sequences defined three groups that were congruent with those defined by CNRE-1 restriction fragment length polymorphisms. PCR amplification of an rDNA intergenic spacer revealed conservation of the intergenic spacer length within groups. Electrophoretic karyotyping did not distinguish between two isolates in each of two CNRE-1 groups. DNA from all isolates studied hybridized to an alpha mating type-specific probe. We interpret these results as suggesting a clonal population structure for some pathogenic isolates of C. neoformans in New York City.

Structure of the ubiquitin-encoding genes of Cryptococcus neoformans.

Cryptococcus neoformans (Cn) contains two ubiquitin (UBI)-encoding genes located on separate chromosomes. The UBI1 gene consists of UBI fused to a 53-amino-acid (aa) tail and is 95% identical to the Saccharomyces cerevisiae (Sc) UBI1 which codes for an UBI-CEP52 ribosomal protein fusion. UBI4 is a polyubiquitin gene that contains five UBI repeats. The UBI4 aa sequences differ from Sc UBI by a single aa. UBI1 contains two introns in the UBI-encoding portion and two introns in the tail. Single introns are present in three of the repeats in UB14 and are located at the same positions as those in UBII. There was also an average of 15% nt differences among UBI repeats. The results provide evidence of extensive recombination and/or conversion events between repeated genes in Cn.

Variation in the structure of glucuronoxylomannan in isolates from patients with recurrent cryptococcal meningitis.

Capsular glucuronoxylomannans (GXM) of Cryptococcus neoformans var. neoformans isolates from patients with recurrent cryptococcal meningitis were analyzed by 1H nuclear magnetic resonance spectroscopy and for reactivity with factor sera (Iatron, Tokyo, Japan). For each patient the initial and relapse isolates had previously been shown to be indistinguishable by DNA restriction fragment length polymorphism analysis. For patients J11 and J22 the GXM of the initial and relapse isolates were identical. For patients SB4 and SB6 the GXM of the initial and relapse isolates differed in structure and reactivity with factor sera. In patient SB4 the initial isolate had a serotype A/D structure, and the first relapse isolate had a serotype A structure. The second relapse isolate was a mixture of structures composed of serotype D components, glucuronomannan (GM), and a minor serotype A component. Analysis of the initial isolate from patient SB6 showed a structure composed mainly of serotype D, GM, and minor serotype A components and components not assigned to a particular serotype (N). The relapse isolate had the same composition as the initial isolate except for an increase in the serotype A component. This increase in the serotype A component of the relapse isolate resulted in a change in the serological specificity from serotype D to serotype A/D. The initial isolate from patient J9 had serotype D and GM structures. The first two relapse isolates had serotype D, N, and GM structures and a minor serotype A component. The third relapse isolate had mainly a serotype D structure. All the J9 isolates reacted only with serotype D-specific factor serum. These results indicate that some isolates obtained from patients with recurrent C. neoformans infections have undergone a change in GXM structure during the course of infection. The modification of GXM structure observed in some relapse isolates is reflected in changed serological properties. The results may have important implications for the design of vaccines and antibody-based therapeutic strategies against C. neoformans.

Prevalence in Cryptococcus neoformans strains of a polysaccharide epitope which can elicit protective antibodies.

Monoclonal antibody (MAb) 2H1 binds to an epitope in the capsule of Cryptococcus neoformans that can elicit protective antibodies. The binding of MAb 2H1 to C. neoformans strains was studied by agglutination, immunofluorescence, and phagocytosis assays. The MAb 2H1 epitope was present in all 21 isolates studied, including those recovered from patients with recurrent infections.

Sterol composition of Cryptococcus neoformans in the presence and absence of fluconazole.

Analysis of the sterol compositions of 13 clinical isolates of the pathogenic yeast Cryptococcus neoformans obtained from five patients with recurring cryptococcal meningitis showed that, unlike Candida albicans, the major sterols synthesized by this yeast were obtusifoliol (range, 21.1 to 68.2%) and ergosterol (range, 0.0 to 46.5%). There was considerable variation in the sterol contents among the 13 isolates, with total sterol contents ranging from 0.31 to 5.9% of dry weight. The isolates from the five patients who had relapses had different total sterol contents and compositions in comparison with those of the pretreatment isolates, indicating either that the sterols had been changed by therapy or that the patients were infected with new isolates with different sterol compositions. Growth of the cryptococcal isolates in the presence of subinhibitory concentrations of fluconazole (0.25x the MIC) significantly altered the sterol content and pattern. The total sterol content decreased in nine isolates and increased in four isolates in response to pretreatment with fluconazole. Fluconazole had no consistent effect on ergosterol levels. In contrast, fluconazole caused a decrease in obtusifoliol levels and an increase in 4,14-dimethylzymosterol levels in all isolates. These results indicate extensive diversity in sterol content, sterol composition, and sterol synthesis in response to subinhibitory concentrations of fluconazole in C. neoformans strains. We propose that fluconazole inhibits the sterol synthesis of C. neoformans by interfering with both 14 alpha-demethylase-dependent and -independent pathways. No correlation between the sterol compositions of C. neoformans isolates and their susceptibilities to fluconazole was found.

Characterization of the CNRE-1 family of repetitive DNA elements in Cryptococcus neoformans.

The pathogenic yeast Cryptococcus neoformans contains 10-20 dispersed repetitive elements that hybridize to clone CNRE-1.0. Screening of a genomic library with probes derived from CNRE-1.0 identified five phages with restriction maps that overlapped CNRE-1.0 and three additional phages that belonged to two distinct groups. Sequencing of internal 3.5-kb SstI fragments from two CNRE-1-like elements revealed 95% homology, as well as a conserved open reading frame. A PCR-RFLP assay was developed that can distinguish different subfamilies of CNRE-1-like elements.

Use of acridinium-ester-labeled DNA probes for identification of mycobacteria in Bactec 13A blood cultures.

AccuProbe tests for mycobacteria (Gen-Probe) cannot be performed directly on Bactec 13A cultures because of interfering substances. This problem can be circumvented by subculturing to Bactec 12B media. Artifactual chemiluminescence greater than the positive cutoff was seen in 15 of 19 13A cultures containing Mycobacterium avium complex compared with 0 of 19 in 12B subcultures. Of the subcultures, 89% were tested after overnight incubation.

Susceptibilities of serial Cryptococcus neoformans isolates from patients with recurrent cryptococcal meningitis to amphotericin B and fluconazole.

Amphotericin B and fluconazole susceptibilities of 13 Cryptococcus neoformans isolates from five patients with recurrent cryptococcal meningitis were determined. For each patient, serial isolates showed no increase in antibiotic resistance relative to the initial isolate. For these patients, recurrent disease was not due to drug resistance but may reflect changes in immune function and/or poor compliance.

Persistence of initial infection in recurrent Cryptococcus neoformans meningitis.

Patients with cryptococcal meningitis tend to have recurrences of infection. Although the original strain of Cryptococcus neoformans is assumed to persist in recurrent infections, this assumption has not been tested. Southern blot hybridisation with two genomic DNA probes and pulsed-field electrophoresis of intact chromosomes were used to investigate the genetic relation between initial and relapse isolates of C neoformans from patients with recurrent cryptococcal meningitis. Eleven isolates were obtained from four patients (three with AIDS, one with leukaemia). Isolates from each patient could be distinguished from those of the other patients; however, each patient's initial and recurrence isolates were clonally related. Our results provide strong evidence that clinical recurrences of cryptococcal meningitis result from persistence of the original infecting strain.

Rhodamine-auramine O versus Kinyoun-carbolfuchsin acid-fast stains for detection of Cryptosporidium oocysts.

The rhodamine-auramine O stain was compared with the Kinyoun carbolfuchsin acid-fast stain for detection of Cryptosporidium oocysts in samples from patients infected with the human immunodeficiency virus (HIV). A total of 283 fecal specimens from HIV-infected patients were examined for the presence of Cryptosporidium oocysts. Duplicate smears of the fecal concentrates, prepared by the formalin ethyl acetate procedure, were stained by the Kinyoun carbolfuchsin and fluorescent rhodamine-auramine O acid-fast methods. The Kinyoun stain detected 13 positive specimens, while the rhodamine-auramine O stain detected 14 positive specimens. The average time required to survey a stained smear was 2.5 minutes with the fluorescent method, compared with 6.0 minutes with the Kinyoun technique. The rhodamine-auramine O stain is a dependable and efficient method of examining fecal smears for the presence of Cryptosporidium oocysts in a high-risk population.

Use of a dispersed repetitive DNA element to distinguish clinical isolates of Cryptococcus neoformans.

We isolated a recombinant phage from a Cryptococcus neoformans genomic library that contains a member of a dispersed family of repetitive DNA elements. This clone, CNRE-1, hybridized to at least seven chromosomes in C. neoformans on the basis of pulsed-field gel analysis. Hybridization of CNRE-1 to restriction digests of genomic DNA confirmed that there are multiple copies of this element and that restriction fragment length polymorphisms are present in strains from different serotypes of C. neoformans. The utility of this probe as an epidemiologic marker was determined by testing cryptococcal isolates from a single hospital. Five isolates from four patients were closely related to a serotype A reference strain, whereas five other isolates from four additional patients exhibited distinct patterns. In two patients, the isolates obtained during recurrent cryptococcal infections were identical to the original isolates.