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Biomedical subjects

R A Keller

Publications and source records attributed to R A Keller.

At least 19 recordsLinked to original sources
PubMed2001

Hospitalizations for fungal infections after renal transplantation in the United States.

Fungal infections in renal transplant recipients have not been studied in a national population. Therefore, 33,420 renal transplant recipients in the United States Renal Data System from 1 July 1994 to 30 June 1997 were analyzed in a retrospective registry study of hospitalized fungal infections (FI). FI were most commonly associated with secondary diagnoses of esophagitis (68, 23.9%), pneumonia (57, 19.8%), meningitis (23, 7.6%), and urinary tract infection (29, 10.3%). Opportunistic organisms accounted for 95.4% of infections, led by candidiasis, aspergillosis, cryptococcosis, and zygomycosis. Most fungal infections (66%) had occurred by six months post-transplant, but only 22% by two months. In logistic regression analysis, end-stage renal disease due to diabetes, duration of pre-transplant dialysis, maintenance tacrolimus and allograft rejection were associated with FI. In Cox regression analysis, recipients with FI had a relative risk of mortality of 2.88 (95% CI=2.22-3.74) compared to all other recipients. Among FI, zygomycosis and aspergillosis were independently associated with both increased patient mortality and length of hospital stay. Most fungal infections in renal transplant recipients were opportunistic, occurred later than previously reported, and were associated with greatly decreased patient survival. Recipients with diabetes, prolonged pre-transplant dialysis, rejection, and tacrolimus immunosuppression should be considered high risk for FI.

Adolescent↗
PubMed2000

Rapid DNA fingerprinting of pathogens by flow cytometry.

BACKGROUND: A new method for rapid discrimination among bacterial strains based on DNA fragment sizing by flow cytometry is presented. This revolutionary approach combines the reproducibility and reliability of restriction fragment length polymorphism (RFLP) analysis with the speed and sensitivity of flow cytometry. METHODS: Bacterial genomic DNA was isolated and digested with a rare-cutting restriction endonuclease. The resulting fragments were stained stoichiometrically with PicoGreen dye and introduced into an ultrasensitive flow cytometer. A histogram of burst sizes from the restriction fragments (linearly related to fragment length in base pairs) resulted in a DNA fingerprint that was used to distinguish among different bacterial strains. RESULTS: Five different strains of gram-negative Escherichia coli and six different strains of gram-positive Staphylococcus aureus were distinguished by analyzing their restriction fragments with DNA fragment sizing by flow cytometry. Fragment distribution analyses of extracted DNA were approximately 100 times faster and approximately 200,000 times more sensitive than pulsed-field gel electrophoresis (PFGE). When sample preparation time is included, the total DNA fragment analysis time was approximately 8 h by flow cytometry and approximately 24 h by PFGE. CONCLUSIONS: DNA fragment sizing by flow cytometry is a fast and reliable technique that can be applied to the discrimination among species and strains of human pathogens. Unlike some polymerase chain reaction (PCR)-based methods, sequence information about the bacterial strains is not required, allowing the detection of unknown, newly emerged, or unanticipated strains.

DNA Fingerprinting↗
PubMed2000

Flow cytometry-based minisequencing: a new platform for high-throughput single-nucleotide polymorphism scoring.

Single-nucleotide polymorphisms (SNPs) are the most abundant type of human genetic variation. These variable sites are present at high density in the genome, making them powerful tools for mapping and diagnosing disease-related alleles. We have developed a sensitive and rapid flow cytometry-based assay for the multiplexed analysis of SNPs based on polymerase-mediated primer extension, or minisequencing, using microspheres as solid supports. The new method involves subnanomolar concentrations of sample in small volumes ( approximately 10 microl) which can be analyzed at rates of one sample per minute or faster, without a wash step. Further, genomic analysis using multiplexing microsphere arrays (GAMMArrays), enables the simultaneous analysis of dozens, and potentially hundreds of SNPs per sample. We have tested the new method by genotyping the Glu69 variant from the HLA DPB1 locus, a SNP associated with chronic beryllium disease, as well as HLA DPA1 alleles using the multiplexed method. The results demonstrate the sensitivity and accuracy of flow cytometry-based minisequencing, a powerful new tool for genome- and global-scale SNP analysis.

Base Sequence↗
PubMed2000

High-throughput flow cytometric DNA fragment sizing.

The rate of detection and sizing of individual fluorescently labeled DNA fragments in conventional single-molecule flow cytometry (SMFC) is limited by optical saturation, photon-counting statistics, and fragment overlap to approximately 100 fragments/s. We have increased the detection rate for DNA fragment sizing in SMFC to approximately 2000 fragments/s by parallel imaging of the fluorescence from individual DNA molecules, stained with a fluorescent intercalating dye, as they passed through a planar sheet of excitation laser light, resulting in order of magnitude improvements in the measurement speed and the sample throughput compared to conventional SMFC. Fluorescence bursts were measured from a fM solution of DNA fragments ranging in size from 7 to 154 kilobase pairs. A data acquisition time of only a few seconds was sufficient to determine the DNA fragment size distribution. A linear relationship between the number of detected photons per burst and the DNA fragment size was confirmed. Application of this parallel fluorescence imaging method will lead to improvements in the speed, throughput, and sensitivity of other types of flow-based analyses involving the study of single molecules, chromosomes, cells, etc.

Bacteriophage M13↗
PubMed1999

Characteristics of different nucleic acid staining dyes for DNA fragment sizing by flow cytometry.

An efficient and reliable double-stranded DNA (dsDNA) staining protocol for DNA fragment sizing by flow cytometry is presented. The protocol employs 0.8 microM of PicoGreen to label a wide range of DNA concentrations (0.5 ng/mL to 10,000 ng/mL) without regard to the solution dye/bp ratios and without initial quantification of the DNA analyte concentration. Using a combination of spectrofluorometry and flow cytometry experiments, we found that PicoGreen exhibited better overall performance than all the tested dsDNA binding dyes, such as TOTO-1. Fluorometric titration revealed that typical DNA staining protocols designed on the basis of the dye/bp ratio were highly dependent upon the DNA concentration for optimal results. PicoGreen was the least sensitive to the solution dye/bp ratio and was highly fluorescent in the presence of dsDNA. Using this new protocol, accurate histograms of HindIII digested lambda DNA were demonstrated for DNA concentrations ranging from 5 to 2000 ng/mL, and for dye/bp ratios from 106:1 to 1:4 at 0.8 microM of PicoGreen. The new one-step protocol is broadly applicable to any sensitive, laser-induced fluorescence method for detection of nucleic acids.

Coloring Agents↗
PubMed1999

Bacterial fingerprinting by flow cytometry: bacterial species discrimination.

BACKGROUND: A flow cytometric measurement (FCM) technique has been developed to size DNA fragments. Individual fragments of a restriction digest of genomic DNA, stained with an intercalating dye, are passed through an ultrasensitive cytometer. The measured fluorescence intensity from each fragment is proportional to the fragment length. METHODS: The isolation of bacterial genomic DNA and digestion by restriction enzymes were performed inside an agarose plug. Rare cutting enzymes were employed to produce a manageable number of DNA fragments. Electroelution was used to move the DNA fragments from the agarose plug into a solution containing polyamines to protect the DNA from shear-induced breakage. The DNA was stained with the bisintercalating dye thiazole orange homodimer and introduced into our ultrasensitive flow cytometer. A histogram of the fluorescence intensities (fingerprint) was constructed. RESULTS: Gram-positive Bacillus globigii and gram-negative bacteria Escherichia coli and Erwinia herbicola were distinguished by the fingerprint pattern of restriction fragments of their genomic DNA. DNA sizes determined by FCM are in good agreement with pulsed-field gel electrophoresis (PFGE) analysis. Flow cytometry requires only picogram quantities of purified DNA and takes less than 10 min for data collection and analysis. When the total sample preparation time is included, the analysis times for PFGE and FCM are similar ( approximately 3 days). CONCLUSIONS: FCM is an attractive technique for the identification of bacterial species. It is more sensitive and potentially much faster than PFGE.

Bacteria↗
PubMed1999

Bacteria genome fingerprinting by flow cytometry.

BACKGROUND: A flow cytometry-based, ultrasensitive fluorescence detection technique has been developed that demonstrates unique advantages in the analysis of large DNA fragments over the currently most widely used technology, pulsed-field gel electrophoresis (PFGE). The technique described herein is used to characterize the restriction fingerprints of the bacteria genome Staphylococcus aureus in this study. METHODS: The isolation of the bacterial genomic DNA and the subsequent complete digestion by a restriction endonuclease were performed inside an agarose plug. Electroelution was used to move the DNA fragments out-of the agarose plug into a solution containing low concentrations of spermine and spermidine, added to stabilize the large DNA fragments. DNA was stained with the bisintercalating dye thiazole orange homodimer (TOTO-1) and subsequently introduced into our ultrasensitive flow cytometer from a capillary. RESULTS: Individual DNA fragments up to 351 kbp were successfully handled and sized. The histograms of the burst sizes were generated from signals associated with individual fragments in <7 min with <2 pg of DNA. The sizing accuracy was better than 98%. In contrast, standard PFGE takes approximately 20 h and requires approximately 1 microg of DNA with a sizing accuracy of approximately 90%. CONCLUSIONS: With the demonstrated success and advantages, our approach has the potential of being applied to fast, accurate bacteria species and strain identification.

Bacterial Typing Techniques↗
PubMed1999

The dermatologist in military operations.

The military dermatologist has a specific and significant role in military operations--in time of war as well as in peace. Many dermatologists are unfamiliar with the impact that our specialty and cutaneous disease has upon the ability of the military to fulfill the missions, duties, and responsibilities assigned by our government. This article highlights a few of the recent or ongoing types of military operations in which our specialty plays a prominent part.

Cuba↗
PubMed1998

Detection system for reaction-rate analysis in a low-volume proteinase-inhibition assay.

High-throughput screening of large combinatorial chemical libraries in biochemical assays will benefit from reduced reagent volume and increased speed of measurement. Standard assays typically are performed in 96-well microtiter plates having 200-microL well volumes and up to an hour of incubation time. In this paper, we demonstrate a technique for precise and rapid measurement of the progress of an enzymatic reaction and its inhibition with reduced volume and time (for this work, the assay was mixed at the 200-microL level and detected in 2-microL volumes with minutes of total assay time). Directly measuring the enzyme activity in the small volume format yields a precise value for the median inhibitory concentration (IC50) of an inhibitor compound. The model assay is the endoproteolytic cleavage of a small fluorogenic peptide by human neutrophil collagenase (MMP-8). The fluorogenic peptide was labeled at one end with a UV/blue fluorophore (N-methylanthranilyl) and at the other end with a quencher (dinitrophenol). To generate inhibition data, a hydroxamate peptide analog inhibitor of collagenase, actinonin, was included in the reaction. The experiments were performed using ultraviolet laser illumination (325 nm wavelength) and parallel fluorescence detection by a cooled, charge-coupled-device camera system to increase sensitivity and speed. The assay volume was reduced to 2 microL for data collection, and the total time for mixing, incubation, and measurement was less than 6 min. For comparison to a standard format, the same assay was performed in a 96-well microtiter plate in 200 microL using 30 min of incubation and measurement in a microtiter plate fluorimeter. Median inhibitory concentrations (IC50) for actinonin of 73 +/- 16 and 100 +/- 14 nM were obtained in the 2- and 200-microL assays, respectively. One concern with assay miniaturization and increases in throughput is a potential loss of precision and accuracy. Laser excitation and parallel detection of fluorescence is a promising approach for increased speed and reduced cost without loss of precision for proteinase inhibition assays.

Fluorescence↗
PubMed1997

New flow cytometric technologies for the 21st century.

The envelope that defines the limits within which flow cytometry was developed is being rapidly expanded. For example: detection sensitivity has been extended to single molecules, the size range of "particle" analysis now extends from DNA fragments to plankton (1,000.+ microns), cell and chromosome sorting rates are being increased dramatically by using inactivation procedures (50,000 per second versus 2,000 per second), rapid kinetic flow cytometry enables real-time analysis of molecular assembly and cell function in the sub-second time domain, the lifetime of a fluorochrome bound to a single cell can be measured with nsec precision, and classical karyotype information (cell to cell heterogeneity) can be determined in a flow based system. These frontiers have greatly expanded the range of new and exciting flow cytometric based biomedical applications. New enabling technologies have provided the means to measure DNA cleavage by the structure-specific nuclease, human Flap Endonuclease (FEN-1), in the 300 msec time frame. Phase sensitive measurements and fluorescence lifetime are proving to be major advances for understanding molecular environments that change with, for example, the process of apoptosis. The ability to detect single fluorescent molecules has been applied to the analysis of DNA fragments obtained from enzymatic digestion of lambda DNA. This technology is being used to rapidly and very accurately size DNA fragments for the human genome project. Optical chromosome selection is a faster, better, less complex approach to chromosome sorting. This method is based on the induction of specific damage to the DNA of selected chromosomes. Lastly, the miniaturization of a single cell fractionator has made it possible to perform single cell flow cytogenetics.

Chromosomes, Human↗
PubMed1997

Rickettsialpox-like illness in a traveler.

A patient presented after a trip to South Africa with a febrile illness and rash that was consistent with either rickettsialpox or mild boutonneuse fever. The clinical, laboratory, and geographic overlap of these diseases makes differentiation difficult in certain situations. Several different rickettsial infections may cause an eschar and a rash that may be papulovesicular. From a clinical perspective, distinguishing these diseases is not critically important as long as therapy with tetracycline is implemented. More precise identification of the etiologic agent could be required in certain military situations because the preventive measures employed for some of these diseases may be significantly different.

Boutonneuse Fever↗
PubMed1996

Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones.

A flow cytometry-based, ultrasensitive fluorescence detection technique is used to size individual DNA fragments up to 167 kb in length. Application of this technology to the sizing of P1 artificial chromosomes (PACs) in both linear and supercoiled forms is described. It is demonstrated that this method is well suited to characterizing PAC/BAC clones and will be very useful for the analysis of large insert libraries. Fluorescence bursts are recorded as individual, dye stained DNA fragments pass through a low power, focused, continuous laser beam. The magnitudes of the fluorescence bursts are linearly proportional to the lengths of the DNA fragments. The histograms of the burst sizes are generated in <3 min with <1 pg of DNA. Results on linear fragments are consistent with those obtained by pulsed-field gel electrophoresis. In comparison with pulsed-field gel electrophoresis, sizing of large DNA fragments by this approach is more accurate, much faster, requires much less DNA, and is independent of the DNA conformation.

Chromosomes↗
PubMed1996

Cutaneous herpes simplex virus, type I, in association with Staphylococcus aureus in an infant.

A 9-month-old infant was diagnosed as having impetigo of the central face. Her clinical condition deteriorated despite treatment with intravenous antibiotics. Viral and bacterial cultures grew herpes simplex virus type I and Staphylococcus aureus, respectively. The patient's condition improved rapidly with antiviral treatment in combination with antibiotics. Recognition of the possibility of a combined viral and bacterial infection is important so that adequate treatment is not delayed.

Facial Dermatoses↗
PubMed1995

Spatial dependence of the optical collection efficiency in flow cytometry.

The sensitive flow cytometric detection of fluorescent species in liquid sample streams requires efficient collection of light from small [approximately 1 picoliter (pl)] sample volumes. This is often accomplished with high numerical aperture (NA) imaging collection optics used in combination with a spatial filter. A method to measure the spatial variation of the optical collection efficiency within the sample volume, using a submicrometer light source, is described. Measurements of the relative optical collection efficiency are presented for two optical collection systems used in our laboratory for single molecule detection. The measurement are in qualitative agreement with relative optical collection efficiency calculations using a simple geometrical optics model. Absolute measurements of the peak collection efficiencies for the two collection systems are also presented. These absolute collection efficiency measurements are in good quantitative agreement with ideal collection efficiencies calculated using measured transmissions and rated NAs of the collection optics. The utility of this information for the characterization and optimization of sensitive fluorescence detection apparatus is discussed.

Equipment Design↗
PubMed1995

Surgical wound healing complicated by Aspergillus infection in a nonimmunocompromised host.

BACKGROUND: An unusual complication of cutaneous surgery and its management is presented. Aspergillus flavus was identified from a nonhealing surgical wound of the ear undergoing cartilaginous necrosis in an immunocompetent host. OBJECTIVE: We wish to alert clinicians that Aspergillus may infect surgical wounds of the ear causing significant morbidity. METHOD: A healthy man underwent Mohs micrographic surgery for invasive Bowen's disease of the ear. Due to the size and location of the defect it was allowed to heal by secondary intent. The patient developed inflammation and subsequent destruction of the ear cartilage. Aspergillus was demonstrated by touch preps and cultured from swabs and tissue from the necrotic wound. RESULTS: In spite of aggressive topical and oral antifungal therapy severe distortion of the pinna occurred, resulting in surgical removal of the upper two-thirds of the ear. CONCLUSIONS: In the presence of cartilage necrosis following surgery on the ear, Aspergillus infection should be considered. Early aggressive management with surgical debridement, and topical and oral antifungal therapy may prevent destruction of the cartilage and a significant cosmetic defect.

Aspergillosis↗