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Biomedical subjects

K E Willard

Publications and source records attributed to K E Willard.

At least 19 recordsLinked to original sources
PubMed1992

Nonparametric probability density estimation: improvements to the histogram for laboratory data.

The histogram has long been used in the clinical laboratory for the depiction and manipulation of frequency data. We present recent results of refinements to the usual histogram procedures along with modern alternative methods of estimating frequency distributions, including the kernel and discrete maximum penalized likelihood estimation (DMPLE) approaches. We compared these nonparametric methods on 15 different types of simulated distributions, and on several sets (greater than 1000 subjects/set) of real data, including alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase levels. Each frequency curve estimation technique was evaluated by measuring the integrated mean square error between each technique's prediction and the true underlying distribution, using Monte Carlo techniques on sample sets with size 49 and 119. The kernel method was the clear method of choice, both in performance (best in 22/36 cases) and in practical usage.

Alanine Transaminase↗
PubMed1992

Detection of gyrA gene mutations associated with ciprofloxacin resistance in methicillin-resistant Staphylococcus aureus: analysis by polymerase chain reaction and automated direct DNA sequencing.

A portion of the gyrA gene from amino acid codons 67 to 129 was sequenced in 34 methicillin-resistant Staphylococcus aureus strains (14 isolated in Minnesota, 10 isolated in Indiana, and 10 isolated in Tennessee). Twenty-eight of these strains were ciprofloxacin resistant. Sixteen of the strains contained a Ser----Leu mutation at codon 84; 3 contained strains a Ser----Ala mutation at codon 84; 3 strains contained two mutations, Ser----Leu at codon 84 and Ser----Pro at codon 85; and 6 strains contained a Glu----Lys mutation at codon 88. Six strains were wild type and ciprofloxacin susceptible. Several mutations from amino acid codons 84 through 88 can be associated with high-level quinolone resistance.

Base Sequence↗
PubMed1992

Comparison of the VIDAS Clostridium difficile toxin A immunoassay with C. difficile culture and cytotoxin and latex tests.

The VIDAS Clostridium difficile toxin A immunoassay (CDA) is a new, automated, enzyme-linked fluorescent-antibody assay for detection of C. difficile toxin A antigen in stool specimens. Simultaneous, parallel testing was performed by using the VIDAS CDA, the Culturette brand CDT latex test for C. difficile antigens, and conventional laboratory cell culture tests for C. difficile, cytotoxicity and C. difficile culture. One hundred ninety-four consecutive fresh soft or liquid stool samples submitted for C. difficile testing between July and September 1990 were evaluated. Of the 194 samples tested, 19 (10%) were from 16 patients who met our case definition for C. difficile-associated disease. The in vitro tests were evaluated in relation to two forms of a clinical case definition. In one form, a positive culture for toxin-producing C. difficile or a positive cytotoxin result obtained directly from the stool specimen was required as laboratory evidence of C. difficile. In the other, a positive result of any of the four laboratory tests was accepted for the laboratory portion of the case definition. No significant difference between the sensitivity of the VIDAS CDA and that of the Culturette brand CDT latex test was found (48 to 58% sensitivity for the CDT latex test and 52 to 63% sensitivity for the VIDAS CDA compared with 93 to 100% sensitivity for culture and 70 to 100% sensitivity for cytotoxin testing). The performance of the VIDAS CDA, however, was hampered by a high percentage of tests (19%) which gave an uninterpretable result.

Bacterial Toxins↗
PubMed1991

A general ampC active site oligonucleotide probe for gram-negative rods.

We have developed a 33 mer probe that hybridizes to the serine active site of the chromosomal ampC beta-lactamase gene of Pseudomonas aeruginosa and the Enterobacteriaceae. We tested this probe against a variety of Enterobacteriaceae, and a series of 23 P. aeruginosa by dot-blots and selected Southern blots. This probe is an alternative or supplement to enzyme studies for characterizing the class of a Gram-negative rod's beta-lactamase and is a useful tool for studies of Pseudomonas beta-lactamase regulation.

Base Sequence↗
PubMed1991

An evaluation of three commercial fecal transport systems for the recovery of enteric pathogens.

Twenty-five isolates, including six strains of Shigella species, six strains of Salmonella species, five strains of Yersinia enterocolitica, six strains of Campylobacter jejuni, and two strains of Vibrio parahaemolyticus, were inoculated at a concentration of 1.5 x 10(4) colony-forming units/mL into the following transport systems: Fekal Enteric Plus (Trend Scientific, Inc., St. Paul, MN), Cary Blair Transport Medium (Remel, Inc., Lenexa, KS), and Para-Pak C & S (Meridian Diagnostics, Inc., Cincinnati, OH). The Fekal Enteric Plus system showed a greater than or equal to 50% recovery of the original bacterial inoculum after 96 hours for all Salmonella, Shigella, and Yersinia strains tested and after as long as 72 hours for Vibrio strains. The Cary Blair Transport System showed greater than or equal to 50% recovery of the initial inoculum at 96 hours for five of six Salmonella strains, four of six Shigella strains, all Yersinia strains, and one of two Vibrio strains. With the use of the Para-Pak C & S, greater than or equal to 50% recovery of the original inoculum after 96 h was demonstrated for five of six Salmonella strains, four of six Shigella strains, all Yersinia strains, and one of two Vibrio strains. All three systems did not demonstrate even 10% recovery of the initial C. jejuni inoculum at 24 hours when held at room temperature.

Bacteriological Techniques↗
PubMed1991

The slide centrifuge gram stain as a urine screening method.

A slide centrifuge Gram stain procedure was performed to screen for bacteriuria 4161 urine specimens submitted in urine preservative tubes for routine culture. For slide centrifuge Gram staining, each urine sample was mixed well. Thereafter, 0.2 mL of each sample was placed, using a pipette, into a slide centrifuge chamber and centrifuged at 2,000 rpm for 5 minutes. The slides were heat fixed, Gram stained, and read by laboratory personnel who scanned 12 consecutive oil-immersion fields using a set pattern. The presence of the same organism in six or more fields was defined as a positive urine screen. Urine samples were cultured using a 0.001-mL loop and a comparison of culture growth with slide centrifuge screening was made. When growth of 100,000 or more colony-forming units per milliliter (CFU/mL) was the reference for comparison, the screen had a sensitivity rate of 98%, a specificity rate of 90%, a negative predictive value of 99%, and a positive predictive value of 65%. When a lower colony count of 10,000 or more CFU/mL was the reference for comparison, the screen had a sensitivity rate of 88%, a specificity rate of 95%, a negative predictive value of 96%, and a positive predictive value of 84%. The slide centrifuge Gram stain is a very sensitive screening method to detect bacteriuria in an adult male population.

Adult↗
PubMed1991

Uptake of 3H-norfloxacin in methicillin-resistant Staphylococcus aureus.

To examine the possibility of a proton-motive efflux pump for quinolones in highly quinolone-resistant clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), we studied 3H-norfloxacin uptake in two quinolone-resistant and two quinolone-sensitive strains of MRSA whose gyrA region surrounding amino acid codons 84 and 85 had been sequenced. Two strains were related (one sensitive and one resistant) in that both were recovered from a single patient, one before (sensitive) and one after (resistant) ciprofloxacin therapy. Drug uptake was assessed in four separate experiments running triplicate bacterial suspensions with radiolabeled drug added at time = 0. Sampling was performed in 10 min increments up to 50 min by a vacuum filtration method. The ionic uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCPH), was added at 40 min to test inhibition of a pump mechanism. The results demonstrated no statistically significant differences in uptake between the sensitive and resistant groups, and the uptake patterns were similar. CCCPH also induced an equivalent surge, or enhanced uptake among these strains, rendering an energy-dependent efflux pump an unlikely contributor to the high levels of resistance seen in our strains. Our findings support parallel studies done on these isolates that implicate mutational changes at amino acid codon 84 and/or codon 85 in the gyrA gene as an explanation for high-level quinolone resistance (MIC to ciprofloxacin greater than or equal to 16 mg/L) in MRSA.

Anti-Infective Agents↗
PubMed1991

Comparison of traditional gas chromatography (GC), headspace GC, and the microbial identification library GC system for the identification of Clostridium difficile.

Three gas chromatography (GC) methods were compared for the identification of 52 clinical Clostridium difficile isolates, as well as 17 non-C. difficile Clostridium isolates. Headspace GC and Microbial Identification System (MIS) GC, an automated system which utilizes a software library developed at the Virginia Polytechnic Institute to identify organisms based on the fatty acids extracted from the bacterial cell wall, were compared against the reference method of traditional GC. Headspace GC and MIS were of approximately equivalent accuracy in identifying the 52 C. difficile isolates (52 of 52 versus 51 of 52, respectively). However, 7 of 52 organisms required repeated sample preparation before an identification was achieved by the MIS method. Both systems effectively differentiated C. difficile from non-C. difficile clostridia, although the MIS method correctly identified only 9 of 17. We conclude that the headspace GC system is an accurate method of C. difficile identification, which requires only one-fifth of the sample preparation time of MIS GC and one-half of the sample preparation time of traditional GC.

Bacteriological Techniques↗
PubMed1990

Synthesis and structure-activity relationships of new 9-N-alkyl derivatives of 9(S)-erythromycylamine.

A series of new 9-N-alkyl derivatives of 9(S)-erythromycylamine has been synthesized by reductive alkylation of erythromycylamine with aliphatic aldehydes and sodium cyanoborohydride. Alternative syntheses employing hydrogenation methods have also been developed. These new 9-N-alkyl derivatives possess excellent antimicrobial activity in vitro and in vivo, especially when administered orally to treat experimental infections in mice. From structure-activity studies, 9-N-(1-propyl)erythromycylamine (LY281389) was selected as the most efficacious derivative. These methods have also been extended to the synthesis of some 9-N,N-dialkyl derivatives of erythromycylamine.

Alkylation↗
PubMed1989

Synthesis and antimicrobial evaluation of 20-deoxo-20-(3,5-dimethylpiperidin-1-yl)desmycosin (tilmicosin, EL-870) and related cyclic amino derivatives.

A series of 20-deoxo-20-cyclic (alkylamino) derivatives of tylosin, desmycosin, macrocin and lactenocin was prepared by reductive amination of the C-20 aldehyde group. The majority of the compounds were prepared using metal hydrides (sodium cyanoborohydride or sodium borohydride) as the reducing agents and a suitable cyclic alkylamine. Subsequently, a more convenient procedure was developed using formic acid as a reducing agent. The C-20 amino derivatives prepared from desmycosin exhibited good in vitro antimicrobial activity against Pasteurella haemolytica and Pasteurella multocida (MIC range of 0.78 approximately 6.25 micrograms/ml) as well as Mycoplasma species (MIC range of 0.39 approximately 6.25 micrograms/ml). Several derivatives showed excellent oral efficacy against infections caused by P. multocida in chicks. One of these derivatives, 20-deoxo-20-(3,5-dimethylpiperidin-1-yl)desmycosin (tilmicosin or EL-870) was selected for development as a therapeutic agent for pasteurellosis in calves and pigs.

Amination↗
PubMed1989

Structure-activity studies of 20-deoxo-20-amino derivatives of tylosin-related macrolides.

Reductive amination of the C-20 aldehyde group of tylosin and related macrolides yielded a large series of derivatives with potentially useful antibiotic properties. Evaluation of these new compounds was conducted on the basis of: 1) Broad antimicrobial spectrum in vitro, with particular emphasis on inhibition of Pasteurella multocida and Pasteurella haemolytica; 2) in vivo efficacy, especially when given orally, against P. multocida in experimental infections in chicks; and 3) bioavailability after oral administration to laboratory animals. The most useful activity was found within a series of derivatives produced by reductive amination of desmycosin with secondary amines.

Amination↗
PubMed1989

Synthesis of new analogs of echinocandin B by enzymatic deacylation and chemical reacylation of the echinocandin B peptide: synthesis of the antifungal agent cilofungin (LY121019).

The antifungal antibiotic, echinocandin B (ECB), was modified by a sequential procedure in which the initial step involved enzymatic removal of the native N-linoleoyl group from the N-terminus using an Actinoplanes utahensis culture. The resulting product, ECB nucleus, was reacylated using active esters or acid halides of various substituted acids to give a series of ECB analogs. These analogs possessed anti-Candida activity both in vitro and in vivo (mice). Other studies have shown that one of these, cilofungin, the 4-n-octyloxybenzoyl-ECB analog (LY121019), has excellent anti-Candida activity, low toxicity and is superior to other available antifungal antibiotics.

Acylation↗
PubMed1989

Monte Carlo simulation and the clinical laboratory.

Complex decisions regarding the scope, efficiency, and effectiveness of service coupled with advances in microcomputer workstations and modeling software have created new incentives and opportunities for the application of powerful simulation methodologies in the clinical laboratory. Monte Carlo techniques that involve a large number of repetitive simulations of a system that has probabilistic characteristics may be especially useful. These techniques can be applied to problems where complex interactions, the nondeterministic nature of medical problems, and the incompleteness of medical knowledge render traditional analytic techniques impotent. Examples include complex clinical laboratory data analysis, clinical strategy development using advanced decision analysis methodologies, and prospective evaluation of the effects of proposed changes in laboratory operations. Although Monte Carlo simulation techniques appear to be promising for clinical laboratory use, limitations must be noted. Care and effort are required in model specification if meaningful results are to be obtained and their significance convincingly conveyed.

Computer Simulation↗
PubMed1988

Synthesis and evaluation of tylosin-related macrolides modified at the aldehyde function: a new series of orally effective antibiotics.

Modification of the aldehyde group in tylosin and related macrolide antibiotics dramatically enhanced the oral efficacy of the derivatives against experimental infections caused by susceptible bacteria in laboratory animals. A large number and wide variety of aldehyde-modified macrolide derivatives were prepared, utilizing the Mitsunobu reaction and other chemical transformations. Evaluation of in vitro and in vivo antimicrobial activity indicated that derivatives of demycarosyltylosin (desmycosin) combined the broadest spectrum of antimicrobial activity with the best efficacy and bioavailability after oral administration.

Administration, Oral↗
PubMed1988

In vitro and in vivo evaluation of C-20- and C-23-modified derivatives of tylosin against veterinary pathogens.

Three series of semi-synthetic derivatives of tylosin-related macrolides were evaluated for utility in veterinary medicine. 23-Modified derivatives of 5-O-mycaminosyltylonolide (OMT) possessed potent activity in vitro against species of Pasteurella and Mycoplasma. An experimental infection in chicks caused by Pasteurella multocida was utilized to evaluate efficacy; several of these derivatives of OMT effectively treated the infection when given subcutaneously, but none were effective after oral administration in drinking water. Macrolides retaining the 4'-O-mycarosyl moiety (tylosin, DMT) had relatively poor activity against Pasteurella in vitro. Certain 20-modified derivatives of desmycosin demonstrated good oral bioavailability in chicks and a lead compound with oral efficacy in the Pasteurella infection model was discovered.

Animals↗
PubMed1987

Synthesis, antimicrobial evaluation and structure-activity relationships within 23-modified derivatives of 5-O-mycaminosyltylonolide.

A large series of C-23-modified derivatives of 5-O-mycaminosyltylonolide were synthesized, in which the C-23 hydroxyl group was replaced by halo, aryl ether or thioether, azido, amino or dialkylamino substituents via SN2 displacement reactions. The majority of derivatives possessed excellent in vitro activity against a variety of aerobic and anaerobic bacteria. While some of the compounds treated experimental infections in rodents by parenteral administration, none showed any significant efficacy or bioavailability after oral dosing. Novel rearrangement products were obtained from some of the reactions; these were identified as 13,23-cyclopropyl-12,22-exomethylene and 13,23-cyclopropyl-12-alkoxy derivatives.

Animals↗
PubMed1986

Probabilistic sensitivity analysis methods for general decision models.

Probabilistic sensitivity analysis has previously been described for the special case of dichotomous decision trees. We now generalize these techniques for a wider range of decision problems. These methods of sensitivity analysis allow the analyst to evaluate the impact of the multivariate uncertainty in the data used in the decision model and to gain insight into the probabilistic contribution of each of the variables to the decision outcome. The techniques are illustrated using Monte Carlo simulation on a trichotomous decision tree. Application of these powerful tools permits the decision analyst to investigate the structure and limitations of more complex decision problems with inherent uncertainties in the data upon which the decisions are based. Probabilistic sensitivity measures can provide guidance into the allocation of resources to resolve uncertainty about critical components of medical decisions.

Decision Theory↗