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G D Roberts

Publications and source records attributed to G D Roberts.

At least 37 records · Page 2Linked to original sources
PubMed1998

Histologic parameters predictive of mycobacterial infection.

Tissue specimens from a wide variety of anatomic locations are frequently examined for mycobacteria using a combination of cultures and special stains. Auramine-rhodamine (AR) staining is a sensitive method for detecting acid-fast bacilli (AFB) in tissue sections. We reviewed 85 AR-positive and 275 randomly selected AR-negative biopsy specimens collected during the past 2 years at the Mayo Clinic, Rochester, Minn. Pathologic diagnoses and culture results were also reviewed. Biopsy specimens containing necrotizing granulomas yielded the highest positivity rate for AFB (61 [47.7%]), followed by nonnecrotizing granulomas (14 [17.7%]). Poorly formed granulomas (5 [16.1%]) and acute inflammation (5 [15.6%]) were less frequently positive. Cases with fibrotic or hyalinized granulomas, nonspecific chronic inflammation, nonspecific reactive or reparative changes, no significant histologic abnormality, or malignancy failed to disclose AFB. These specimens, which were consistently negative for AFB, were responsible for 25% of the samples submitted. Of the 360 tissue specimens submitted, 166 had a corresponding mycobacterial culture. Mycobacteria were cultured only from the biopsy specimens that contained necrotizing granulomas (38.2%), nonnecrotizing granulomas (32.4%), poorly formed granulomas (30.0%), or acute inflammation (15.8%). Tissues with fibrotic or hyalinized granulomas, nonspecific chronic inflammation, nonspecific reactive or reparative changes, no significant histologic abnormality, or malignancy failed to yield positive cultures. These data suggest that biopsy specimens with these latter diagnoses are inappropriate specimens for mycobacterial culture or AR staining.

Acute Disease↗
PubMed1998

Clinical evaluation of the Gen-Probe Amplified Direct Test for detection of Mycobacterium tuberculosis complex organisms in cerebrospinal fluid.

Eighty-four cerebrospinal fluid (CSF) samples from different children who presented with signs and symptoms of meningitis were evaluated for the presence of Mycobacterium tuberculosis complex organisms by the Gen-Probe Amplified Mycobacterium tuberculosis Direct Test (MTD; Gen-Probe, San Diego, Calif.). All CSF samples had negative acid-fast smears by the Ziehl-Neelsen staining method. M. tuberculosis was recovered from five samples. M. tuberculosis did not grow from 19 additional samples, but the samples were from patients who fulfilled specific clinical and laboratory criteria for probable tuberculous meningitis (TBM). The remaining samples (n = 60) were from patients with other infections or noninfectious causes of meningitis. The results of the MTD were interpreted as positive or negative on the basis of recommended cutoff values for respiratory specimens. These results were interpreted as true or false positives or true or false negatives on the basis of the results of M. tuberculosis culture or whether the patient fulfilled criteria for probable TBM. The Gen-Probe MTD was 33% sensitive and 100% specific for detecting M. tuberculosis complex organisms in these 84 CSF samples. If the cutoff values for positive results were decreased for the MTD (> or = 11,000 versus > or = 30,000 relative light units), the sensitivity increased to 83% and the specificity remained 100%. These results for the MTD are encouraging considering that TBM is a highly fatal disease and difficult to diagnose by conventional laboratory techniques.

Adolescent↗
PubMed1997

Comparison of agar dilution, broth microdilution, disk diffusion, E-test, and BACTEC radiometric methods for antimicrobial susceptibility testing of clinical isolates of the Nocardia asteroides complex.

An evaluation was undertaken to determine the optimal method for the in vitro susceptibility testing of 26 Nocardia asteroides complex isolates to the following antimicrobial agents: amikacin, ampicillin, amoxicillin-clavulanate, ceftriaxone, ciprofloxacin, erythromycin, imipenem, minocycline, and trimethoprim-sulfamethoxazole. Five testing methods were studied including the agar dilution, broth microdilution, and disk diffusion methods, the epsilometer test (E-test), and the BACTEC radiometric method. Results for each antimicrobial agent and each testing method were interpreted as indicating susceptibility, intermediate susceptibility, or resistance according to current guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) for bacteria that grow aerobically and were then compared to a "gold standard" susceptibility test result. The gold standard result for each Nocardia isolate was established by a consensus of the results of the majority of testing methods used in the study. When the results were combined for all antimicrobial agents tested against all Nocardia isolates by all methods, the BACTEC radiometric method produced the highest level of agreement (97.9%) with the consensus results and had the fewest very major (n = 1), major (n = 2), and minor (n = 2) errors. In contrast, the results of the agar dilution method were in least agreement (93.2%) with the consensus results, and this method also produced the most very major (n = 8), major (n = 4), and, along with the disk diffusion method, minor (n = 6) errors. For all test methods, interpretive errors were most frequent when testing ampicillin or amoxicillin-clavulanate. Moreover, for all Nocardia nova isolates tested, ampicillin susceptibility results by any of the testing methods were not in agreement with the results of testing for beta-lactamase by the nitrocefin (Cefinase) disk method. We conclude that among the methods evaluated, the BACTEC radiometric method appeared to be the best for determining the in vitro susceptibilities of members of the N. asteroides complex to a panel of nine antimicrobial agents. However, none of the test methods, including the BACTEC method, accurately predicted the ampicillin resistance of the N. nova isolates tested, all of which produced beta-lactamase. Presuming that this beta-lactamase hydrolyzes ampicillin, this disparity may relate to the NCCLS breakpoints that were used, which may require modification for this antimicrobial agent when tested against N. nova isolates.

Drug Resistance, Microbial↗
PubMed1997

Clinical comparison of BACTEC 9240 plus aerobic/F resin bottles and the isolator aerobic culture system for detection of bloodstream infections.

The Plus Aerobic/F resin bottle of the BACTEC 9240 automated blood culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) was compared with aerobic culture of the Isolator system (Wampole Laboratories, Cranbury, N.J.) for the detection of bloodstream microorganisms from 6,145 blood cultures collected from adult patients with suspected septicemia. The BACTEC resin bottles were incubated for 7 days, and the sediment from the Isolator tube was inoculated to sheep blood and chocolate agars which were incubated for 72 h and to inhibitory mold, brain heart infusion, and Sabouraud agars which were incubated for 21 days. A total of 622 microorganisms were recovered from 583 blood cultures. The BACTEC resin bottle recovered statistically significantly more pathogens overall than the Isolator system (P = 0.0006). When individual pathogens isolated from either system for a 7-day study period were assessed, it was determined that the BACTEC resin bottle detected statistically significantly more isolates of Staphylococcus aureus (P = 0.0113) and coagulase-negative Staphylococcus spp. (P = 0.0029) than the Isolator system. The BACTEC resin bottle also detected statistically significantly more bloodstream infections (septic episodes) caused by coagulase-negative Staphylococcus spp. (P = 0.0146). The Isolator system recovered statistically significantly more contaminants overall (P < 0.0001), and among this group of microorganisms, recovered statistically significantly more Bacillus spp. (P < 0.0001), coagulase-negative Staphylococcus spp. (P < 0.0001), and viridans group Streptococcus spp. (P = 0.0156). The Isolator system detected statistically significantly more isolates of Histoplasma capsulatum (P = 0.004), but all of these isolates were detected at > or = 7 days of incubation of fungal plates, i.e., after the system to system comparison study period (7 days). In blood culture sets which produced growth of the same pathogen in both systems, there was a statistically significant difference in median time to detection for all pathogens combined favoring the BACTEC resin bottle over the Isolator tube (P < 0.05). When assessing individual microorganisms, the median times for detection of S. aureus, Enterococcus spp., and Pseudomonas spp. were all statistically significantly less for the BACTEC system (P < 0.05). The BACTEC instrument had 79 (1.3%) false positive signals. The BACTEC system required less processing time than the Isolator system and eliminates the hands-on time for detection of positive cultures required with the Isolator system.

Adult↗
PubMed1996

A coordinated high-performance liquid chromatographic, capillary electrophoretic, and mass spectrometric approach for the analysis of oligosaccharide mixtures derivatized with 2-aminoacridone.

Glycans derivatized with 2-aminoacridone have been analyzed consecutively by reverse-phase high-performance liquid chromatography (HPLC) and micellar electrokinetic capillary chromatography (MECC). The 2-aminoacridone derivatizing agent used in the present study is highly hydrophobic and is well separated from the glycan derivatives in both separation techniques, ensuring that excess reagent does not interfere with the oligosaccharide analysis. The methodology outlined uses the high resolving power of capillary electrophoresis to determine the heterogeneity of samples after collection and preconcentration by HPLC. Collected glycan samples are submitted for mass spectrometric analysis to determine molecular weight. This methodology has been applied to linear oligosaccharides derived from dextran and to N-linked mannose-rich glycans from ribonuclease B.

Aminoacridines↗
PubMed1996

Characterization of distinct nuclear and mitochondrial forms of human deoxyuridine triphosphate nucleotidohydrolase.

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase; EC 3.6.1.23) was purified from HeLa cells by immunoaffinity chromatography. Based on SDS-polyacrylamide gel electrophoresis, two distinct forms of dUTPase were evident in the purified preparation. These proteins were further characterized by a combination of NH2-terminal protein sequencing, mass spectrometry, and mass spectrometry-based protein sequencing. These analyses indicate that the two forms of dUTPase are largely identical, differing only in a short region of their amino-terminal sequences. Despite the structural difference, both forms of dUTPase exhibited identical binding characteristics for dUTP. Each form of dUTPase has a distinct cellular localization. Cellular fractionation and isopycnic density centrifugation indicate that the lower molecular weight form of dUTPase (DUT-N) is associated with the nucleus, while the higher molecular weight species (DUT-M) fractionates with the mitochondria. The DUT-N isoform is approximately 30-fold more abundant in HeLa cells than DUT-M as determined by densitometry. The NH2-terminal protein sequence of both DUT-N and DUT-M did not match previous reports of the predicted amino-terminal sequence for human dUTPase (McIntosh, E.M., Ager, D.D., Gadsden, M.H., and Haynes, R.H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8020-8024; Strahler, J.R., Zhu X., Hora, N., Wang, Y.K., Andrews, P.C., Roseman, N.A., Neel, J.V., Turka, L., and Hanash, S.M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4991-4995). A cDNA corresponding to the DUT-N isoform was isolated utilizing an oligonucleotide probe based on the determined NH2-terminal sequence. The cDNA contains a 164-amino acid open reading frame, encoding a protein of Mr 17,748. The DUT-N cDNA sequence matches the previously cloned cDNAs with the exception of a few discrepancies in the 5' end. Our data indicate a 69-base pair addition to the 5' end of the previously reported open reading frame.

Amino Acid Sequence↗
PubMed1996

Evaluation of mass spectrometric methods applicable to the direct analysis of non-peptide bead-bound combinatorial libraries.

Electrospray, matrix-assisted laser desorption, and time-of-flight secondary ion mass spectrometry have been explored as possible methods for the identification of active members of molecular combinatorial libraries. All three methods are found to yield accurate molecular weight information about a target molecule angiotensin II antagonist synthesized on a 40-microns polystyrene bead. Structural identification is also possible by accurate mass measurements to eliminate candidate structures with the same nominal mass and by analysis of the fragmentation patterns. In addition, the secondary ion mass spectrometry measurements yield spatially resolved spectra from a single bead after exposure to a suitable gas which clips the covalent bond at the linking position. All three approaches appear to offer a viable screening strategy of non-peptide libraries without the use of additional molecular tags.

Angiotensin II↗
PubMed1996

Clinical comparison of difco ESP, Wampole isolator, and Becton Dickinson Septi-Chek aerobic blood culturing systems.

The ESP 80A aerobic blood culture of the ESP automated blood culture system (Difco Laboratories. Detroit, Mich.) was compared with two manual aerobic blood culture systems, the Isolator (Wampole Laboratories, Cranbury, N.J.) and the Septi-Chek (Becton Dickinson, Cockeysville, Md.) systems, for the detection of bloodstream microorganisms from 5,845 blood samples for culture collected from adult patients with suspected septicemia. The bottles were incubated for 7 days, and the sediment from the Isolator tube was inoculated onto solid medium and this medium was incubated for 72 h. A total of 609 microorganisms were recovered from 546 blood cultures. There was no statistically significant difference in the total recovery of microorganisms for the ESP 80A system when compared with that for the Septi-Chek system (P = 0.083); however, the Isolator system recovered significantly more microorganisms overall than either the ESP 80A (P < 0.001) or the Septi-Chek (P < 0.001) system. When assessing individual probable pathogens, the Isolator system detected statistically significantly more Staphylococcus aureus and Candida spp. than either the ESP 80A or the Septi-Chek system (P < 0.05). Similarly, the Isolator system detected statistically significantly more bloodstream infections (septic episodes) caused by S. aureus and Candida spp. than either the ESP 80A or the Septi-Chek system (P < 0.05). In blood culture sets which produced growth of the same probable pathogens in the ESP 80A and the Isolator systems, there was no statistically significant difference in the median times to detection for all pathogens combined (P = 0.067). However, a similar comparison showed the Isolator and the ESP 80A systems to have statistically significantly shorter median detection times for all pathogens combined (P < 0.001) when they were independently compared with the Septi-Chek system. The ESP 80A system had 29 (0.5%) false-positive signals. The ESP system required less processing time than the Isolator system and eliminates the hands-on time for the detection of positive cultures required by the manual systems.

Adult↗
PubMed1996

Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods.

Previous studies have indicated that the conventional tests used for the identification of mycobacteria may (i) frequently result in erroneous identification and (ii) underestimate the diversity within the genus Mycobacterium. To address this issue in a more systematic fashion, a study comparing phenotypic and molecular methods for the identification of mycobacteria was initiated. Focus was given to isolates which were difficult to identify to species level and which yielded inconclusive results by conventional tests performed under day-to-day routine laboratory conditions. Traditional methods included growth rate, colonial morphology, pigmentation, biochemical profiles, and gas-liquid chromatography of short-chain fatty acids. Molecular identification was done by PCR-mediated partial sequence analysis of the gene encoding the 16S rRNA. A total of 34 isolates was included in this study; 13 of the isolates corresponded to established species, and 21 isolates corresponded to previously uncharacterized taxa. For five isolates, phenotypic and molecular analyses gave identical results. For five isolates, minor discrepancies were present; four isolates remained unidentified after biochemical testing. For 20 isolates, major discrepancies between traditional and molecular typing methods were observed. Retrospective analysis of the data revealed that the discrepant results were without exception due to erroneous biochemical test results or interpretations. In particular, phenotypic identification schemes were compromised with regard to the recognition of previously undescribed taxa. We conclude that molecular typing by 16S rRNA sequence determination is not only more rapid (12 to 36 h versus 4 to 8 weeks) but also more accurate than traditional typing.

Bacterial Typing Techniques↗
PubMed1996

Prospective evaluation of the utility of molecular techniques for diagnosing nosocomial transmission of multidrug-resistant tuberculosis.

OBJECTIVE: To compare molecular techniques with conventional diagnostic methods for evaluating nosocomial transmission of multidrug-resistant tuberculosis (MDR-TB). DESIGN: We conducted a 12-week postexposure inception cohort study of health-care personnel who had been exposed to a patient with MDR-TB. MATERIAL AND METHODS: In addition to baseline and follow-up tuberculin skin tests and chest roentgenography, weekly pulmonary specimens were evaluated by (1) auramine-rhodamine fluorescent staining, (2) culture for mycobacteria, and (3) polymerase chain reaction (PCR) to amplify IS6110, a nucleic acid insertion sequence unique to the Mycobactrium tuberculosis complex. RESULTS: The index patient's isolate of M. tuberculosis showed a mutation in codon 531 of the RNA polymerase beta subunit (rpoB) gene of M. tuberculosis, which is associated with rifampin resistance and considered a marker for this MDR-TB strain. All pulmonary and gastric specimens from study participants had negative auramine stains and cultures for mycobacteria, One person, however, had separate specimens with repeatedly positive PCR results for IS6110 sequences, but the specimens contained a wild-type M. tuberculosis rpoB codon 531 dissimilar from the index patient's strain. CONCLUSION: Although both molecular and conventional testing showed that no exposed person was infected with the MDR-TB strain, molecular test results were available sooner and seemed more sensitive for detecting M. tuberculosis in one exposed person, presumably in a preinfection or "colonized" stage. Molecular methods provided information that helped distinguish this person's M. tuberculosis strain from the index patient's MDR-TB strain. Additional prospective studies should assess the value of these molecular techniques in similar clinical settings.

Antitubercular Agents↗
PubMed1996

Methods for the rapid identification of mycobacterial species.

The use of rapid tests for the identification of mycobacteria has been advocated primarily for Mycobacterium tuberculosis; however, they have been accepted less widely than expected. Chromatographic methods are best suited for larger laboratories, whereas nucleic acid probes may be used by laboratories that can justify the cost based on volume and pricing. Nucleic acid sequencing offers the possibility of providing the most exact identification of species of mycobacteria, but its use is limited to reference laboratories that have the applicable resources. Because of their clinical importance, all mycobacteria should be identified using the most rapid methods available.

Bacteriological Techniques↗
PubMed1995

An integrated strategy for structural characterization of the protein and carbohydrate components of monoclonal antibodies: application to anti-respiratory syncytial virus MAb.

The relatively rapid and extensive characterization of the amino acid sequence and site-specific carbohydrate structures of a recombinant, reshaped human monoclonal antibody directed against respiratory syncytial virus (RSHZ19) is presented. The integrated strategy used a combination of mass spectrometric and conventional methodologies. Liquid chromatography/electrospray mass spectrometry was used for peptide mapping and selective identification of glycopeptides, and Edman degradation and tandem mass spectrometry were used to define the sequences of selected peptides. Matrix-assisted laser desorption/ionization mass spectrometry provided the M(r) of the intact protein and was used to characterize endo- and exoglycosidase digests of isolated glycopeptides to identify the glycosylation-site peptide and define the structures of the carbohydrates at that site. These experiments verified 99.1% of the light- and 99.3% of the heavy-chain amino acid sequences. The N and C termini of both chains were confirmed, and the nature and extent of heterogeneity at the N and C termini of the heavy chain were determined. Oxidation of a specific methionine residue to the sulfoxide was demonstrated by sequencing the N-terminally blocked peptide by tandem MS. Carbohydrate was found exclusively at Asn296 of the heavy chain. There was no evidence for a nonglycosylated form of the molecule or for the presence of O-linked carbohydrate. The qualitative distribution of glycoforms at this site was determined by MS of the isolated, tryptic glycopeptide and compared with results obtained by high-performance anion exchange chromatography and high-resolution gel permeation chromatography of oligosaccharides released by hydrazinolysis. The sequence and linkage of individual glycan species were determined using matrix-assisted laser desorption/ionization MS to monitor the results of a series of controlled digestions with specific exoglycosidases. The set of glycoforms consists predominantly of biantennary, core fucosylated carbohydrates lacking sialic acid. The present study is one of the first to directly evaluate the quantitative as well as qualitative consistency of the MS methods with conventional methods for carbohydrate analysis.

Amino Acid Sequence↗
PubMed1995

Lag screw stabilization of a cervical vertebral fracture by use of computed tomography in a horse.

A traumatic fracture of C2 was diagnosed radiographically in a 1-year-old German Warm-blood stallion. Fracture configuration was difficult to see on survey radiographs. Computed tomography yielded a more accurate assessment of the fracture and facilitated fracture repair with cortical lag screws. Precise screw placement, to avoid spinal cord damage, was obtained by use of computed tomography. Follow-up radiography revealed normal bone healing, and the horse was in dressage schooling 24 months after surgery.

Animals↗
PubMed1995

Rapid identification of a point mutation of the Mycobacterium tuberculosis catalase-peroxidase (katG) gene associated with isoniazid resistance.

The complete catalase-peroxidase (katG) gene DNA sequence was determined for 15 strains of Mycobacterium tuberculosis with a wide range of susceptibility to isoniazid. Five of 9 strains with isoniazid MICs > or = 1.0 microgram/mL had one or more missense mutations and all 5 strains had a common G-->T transversion in codon 463, causing the replacement of arginine with leucine and the loss of an NciI or MspI restriction site. None of 6 strains with an isoniazid MIC < 1.0 microgram/mL had mutations affecting codon 463. Restriction analysis of 43 strains with isoniazid MICs > or = 1.0 microgram/mL showed that 19 (44.2%) had lost the NciI-MspI restriction site at the locus of codon 463 while only 1 of 32 strains with isoniazid MICs < or = 1.0 microgram/L had this restriction polymorphism. These results indicate that the mutation arginine-->leucine in codon 463 of the catalase-peroxidase gene occurs in a significant fraction (44.2%) of M. tuberculosis strains with isoniazid MICs > or = 1.0 microgram/mL.

Bacterial Proteins↗
PubMed1995

Molecular probes for diagnosis of fungal infections.

We have developed 21 specific nucleic acid probes which target the large subunit rRNA genes from Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus niger, Aspergillus terreus, Blastomyces dermatitidis, Candida albicans, Candida (Torulopsis) glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Coccidioides immitis, Cryptococcus neoformans var. gattii, Cryptococcus neoformans var. neoformans, Filobasidiella neoformans var. bacillispora, Filobasidiella neoformans var. neoformans, Histoplasma capsulatum, Pseudallescheria boydii, and Sporothrix schenckii. A section of the 28S rRNA gene from approximately 100 fungi, representing about 50 species of pathogens and commonly encountered saprophytes, was sequenced to develop universal PCR primers and species-specific oligonucleotide probes. Each step in the process of detection and identification was standardized to a common set of conditions applicable without modification to all fungi of interest and all types of clinical specimens. These steps consist of DNA extraction by boiling specimens in an alkaline guanidine-phenol-Tris reagent, amplification of a variable region of the 28S rRNA gene with universal primers, and amplicon identification by probe hybridization or DNA sequencing performed under conditions identical for all fungi. The results obtained by testing a panel of fungal isolates and a variety of clinical specimens indicate a high level of specificity.

Base Sequence↗
PubMed1995

Direct genotypic detection of Mycobacterium tuberculosis rifampin resistance in clinical specimens by using single-tube heminested PCR.

Recent analysis of the gene encoding the beta subunit of Mycobacterium tuberculosis RNA polymerase (rpoB) has demonstrated a small region that harbors the mutations most frequently associated with rifampin resistance. Earlier reports have described a high degree of sequence conservation of rpoB among mycobacteria other than M. tuberculosis and other GC-rich bacteria that can lead to false-positive amplification when applied directly to clinical specimens. We developed reagents for PCR amplification that are based on signature nucleotides discovered by comparative sequence analysis of the rpoB genes of organisms phylogenetically related to M. tuberculosis. The specificities of the reagents were challenged with 20 isolates of multiple-drug-resistant M. tuberculosis and more than 20 species of mycobacteria other than M. tuberculosis and other GC-rich organisms. A single-tube heminested PCR protocol was devised to obtain sensitivity equal to those of an IS6110-based PCR assay and culture in spiked sputum experiments. The assay correctly identified 21 of 24 (87.5%) culture-positive specimens, 13 of which were acid-fast smear-negative, in a panel of 51 clinical specimens. Three specimens that were false-positive initially were negative upon repeat testing when the assay was modified to eliminate the potential for aerosol carryover of the first-round amplification product during the open-tube addition of the second set of reaction reagents. This assay is the most sensitive and specific test to date for the direct detection of M. tuberculosis rpoB in clinical specimens. This rapid PCR-based assay can be used for the simultaneous identification of M. tuberculosis and its rifampin susceptibility genotype.

Amino Acid Sequence↗