Continuous axenic cultivation of Pneumocystis carinii.
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Continuous axenic culture of Pneumocystis carinii has been achieved. A culture vessel is used that allows for frequent medium exchange without disturbance of organisms that grow attached to a collagen-coated porous membrane. The growth medium is based on Minimal Essential Medium with Earle's salt supplemented with S-adenosyl-L-methionine, putrescine, ferric pyrophosphate, N-acetyl glucosamine, putrescine, p-aminobenzoic acid, L-cysteine and L-glutamine, and horse serum. Incubation is in room air at 31 degrees C. The pH of the medium begins at 8.8 and rises to approximately 9 as the cells grow. Doubling times calculated from growth curves obtained from cultures inoculated at moderate densities ranged from 35 to 65 hours. With a low-density inoculum, the doubling time is reduced to 19 hours. The morphology of cultured organisms in stained smears and in transmission electron micrographs is that of P. carinii, and P. carinii-specific mAbs label the cultured material. Cultured organisms are infective for immunosuppressed rats and can be stored frozen and used to reinitiate culture. (+info)
Species identification and strain differentiation of dermatophyte fungi by analysis of ribosomal-DNA intergenic spacer regions.
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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. (+info)
Aspergillus meningitis: diagnosis by non-culture-based microbiological methods and management.
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The performance of antibody detection, antigen detection, and Aspergillus genus-specific PCR for diagnosing Aspergillus meningitis was investigated with 26 cerebrospinal fluid (CSF) samples obtained from a single patient with proven infection caused by Aspergillus fumigatus. Immunoglobulin G antibodies directed against Aspergillus were not detected by enzyme-linked immunosorbent assay in CSF or serum. The antigen galactomannan was detected in the CSF 45 days before a culture became positive, and Aspergillus DNA was detected 4 days prior to culture. Decline of the galactomannan antigen titer in the CSF during treatment with intravenous and intraventricular amphotericin B and intravenous voriconazole corresponded with the clinical response to treatment. (+info)
A sandwiched-culture technique for evaluation of heterologous protein production in a filamentous fungus.
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Aspergillus niger is known for its efficient excretion machinery. However, problems have often arisen in obtaining high amounts of heterologous proteins in the culture medium. Here we present a quick method using sandwiched colonies to evaluate transgenic strains for secretion of heterologous proteins. Expressing the ABH1 hydrophobin of Agaricus bisporus in A. niger, we showed that low production levels of the heterologous protein are probably due to extracellular proteolytic degradation of the protein. (+info)
Controlled clinical comparison of bioMerieux VITAL and BACTEC NR-660 blood culture systems for detection of bacteremia and fungemia in adults.
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A total of 9,446 blood cultures were collected from adult patients at three university-affiliated hospitals. Of these, 8,943 cultures were received with both aerobic bottles filled adequately; 885 yielded 1,016 microorganisms, including 622 isolates (61%) that were the cause of sepsis, 337 isolates (33%) that were contaminants, and 57 isolates (6%) that were indeterminate as the cause of sepsis. With the exception of Staphylococcus aureus, which was recovered more often from VITAL aerobic bottles, more pathogenic microorganisms were recovered from BACTEC NR6 (aerobic) bottles than from VITAL aerobic bottles. Growth of pathogenic microorganisms was detected earlier in VITAL aerobic bottles. A total of 8,647 blood cultures were received with both anaerobic bottles filled adequately; 655 yielded 740 microorganisms, including 486 isolates (66%) that were the cause of sepsis, 215 isolates (29%) that were contaminants, and 39 isolates (6%) that were indeterminate as the cause of sepsis. More pathogenic microorganisms were recovered from VITAL anaerobic bottles than from BACTEC NR7 (anaerobic) bottles. Growth of pathogenic microorganisms was detected earlier in VITAL anaerobic bottles. In 8,500 sets all four bottles were received adequately filled. When paired aerobic and anaerobic bottle sets (systems) were compared, more pathogenic microorganisms (again with the exception of S. aureus) were recovered from the BACTEC system. For the 304 septic episodes (253 unimicrobial and 51 polymicrobial), significantly more were detected by the BACTEC system. We conclude that VITAL requires modification to improve recovery of pathogenic microorganisms to make it competitive with other commercially available blood culture systems. (+info)
PCR-restriction enzyme analysis for detection of Candida DNA in blood from febrile patients with hematological malignancies.
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Blood samples were drawn daily from 72 patients who had hematological malignancies, neutropenia, and fever and who had failed to respond to broad-spectrum antibiotics. Each sample was used for conventional fungal blood cultures and for detection and identification of Candida DNA by a PCR method with subsequent restriction enzyme analysis (REA) recently developed in our laboratory. The PCR method was able to detect five CFU of Candida spp. per ml of blood, and subsequent REA of the amplicons allowed the identification of the Candida species most commonly implicated in cases of candidiasis. Thirty-one patients were PCR-REA positive, and four of these patients were also culture positive. The ultimate diagnosis for 13 of these patients and 1 patient who was PCR-REA negative was disseminated candidiasis (confirmed by clinical data, multiple cultures, histology, autopsy, and/or ultrasonographic evidence of hepatosplenic candidiasis). The molecular method is significantly more sensitive than conventional fungal blood cultures and has a high negative predictive value (97.5%) for the development of disseminated candidiasis in neutropenic patients. (+info)
Comparison of three commercial systems for identification of yeasts commonly isolated in the clinical microbiology laboratory.
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We evaluated three commercial systems (RapID Yeast Plus System; Innovative Diagnostic Systems, Norcross, Ga.; API 20C Aux; bioMerieux-Vitek, Hazelwood, Mo.; and Vitek Yeast Biochemical Card, bioMerieux-Vitek) against an auxinographic and microscopic morphologic reference method for the ability to identify yeasts commonly isolated in our clinical microbiology laboratory. Two-hundred one yeast isolates were compared in the study. The RapID Yeast Plus System was significantly better than either API 20C Aux (193 versus 167 correct identifications; P < 0.0001) or the Vitek Yeast Biochemical Card (193 versus 173 correct identifications; P = 0.003) for obtaining correct identifications to the species level without additional testing. There was no significant difference between results obtained with API 20C Aux and the Vitek Yeast Biochemical Card system (P = 0.39). The API 20C Aux system did not correctly identify any of the Candida krusei isolates (n = 23) without supplemental testing and accounted for the major differences between the API 20C Aux and RapID Yeast Plus systems. Overall, the RapID Yeast Plus System was easy to use and is a good system for the routine identification of clinically relevant yeasts. (+info)
Times to detection of bacteria and yeasts in BACTEC 9240 blood culture bottles.
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A 7-day incubation protocol was instituted with the BACTEC 9240 system for a 1-year period to determine the times to detection of clinically relevant organisms. A total of 23,686 blood and 693 sterile body fluid cultures were received; some cultures were held longer by special request. Of 1,609 likely skin contaminants, 42 were recovered on day 5, 34 on day 6, 16 on day 7, and 5 on day 8. Of 2,803 usual pathogens, 34 were recovered on day 5, 24 on day 6, 15 on day 7 and 1 on day 8. Twenty-one of the latter organisms were considered significant laboratory isolates because they were the first isolates from the respective patients. Chart review showed that 10 of 21 were considered clinically significant, but only 3 (all yeasts) affected the treatment of the patient. Our data show that 4 days of incubation were sufficient to recover all clinically relevant bacteria and 6 days were required to recover all clinically relevant yeasts. (+info)