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C J Shanholtzer

Publications and source records attributed to C J Shanholtzer.

30 records · Page 2Linked to original sources

Laboratory quality assurance testing of microbiologic media from commercial sources.

Culture media that perform as intended are crucial to accurate work by a clinical microbiology laboratory. As cost-containment becomes an increasingly important pressure in clinical laboratory management, the value of each laboratory performing its own quality assurance testing of media has been challenged if the media are purchased from a manufacturer who is required to perform quality assurance testing. In the past ten years of using commercially prepared media from eight different national and regional suppliers, the authors have encountered an average of six types (15 shipments) of media each year that failed to meet their laboratory's performance criteria, which reinforces the need for clinical laboratories to continue their own performance testing of media manufactured commercially, particularly when the media are used for primary isolation. Quality control testing of media can be performed most cost-effectively by purchasing in the largest lots and shipments that shelf life and storage requirements allow, streamlining test procedures while keeping detailed records of performance characteristics, and purchasing media from firms that have demonstrated competence and integrity.

Bacteria↗

False susceptible penicillin G MICs for Streptococcus pneumoniae with a commercial microdilution system.

A commercial minimal inhibitory concentration (MIC) panel that contains a broth of undisclosed formulation intended to support growth of streptococci unable to grow in Mueller-Hinton broth was found to give false susceptible MIC results for pneumococci. The authors encountered several Streptococcus pneumoniae isolates that showed resistance by the oxacillin disk diffusion test, but showed a susceptible penicillin G MIC when tested in an unsupplemented MIC panel. Seven isolates that showed penicillin G resistance by oxacillin disk diffusion testing were retested for penicillin G susceptibility with oxacillin disk diffusion, commercial MIC panels, Mueller-Hinton with blood agar dilution, and Mueller-Hinton with blood broth microdilution. Five of these isolates produced a button of growth in the commercial MIC panel growth control well, but no growth in the penicillin G-containing wells (0.06-4.0 micrograms/mL), suggesting a valid test system and susceptibility to penicillin G. When tested by the three standard methods, these seven isolates showed resistance (R) or relative resistance (I) to penicillin G. These data indicate that frozen MIC panels from this commercial source (American Micro Scan, Campbell, CA) are not reliable for detection of resistance to penicillin G in S. pneumoniae, and if these panels are used, results must be confirmed by another method.

Evaluation Studies as Topic↗

Detection of Clostridium difficile toxins A (enterotoxin) and B (cytotoxin) in clinical specimens. Evaluation of a latex agglutination test.

A new latex test, Culturette Brand Rapid Latex Test for detection of Clostridium difficile toxin A, was tested on 408 stool samples. In 247 frozen tissue culture supernate specimens previously obtained from patients with C. difficile-associated diarrhea (CAD), the latex test (enterotoxin) was positive in 182 (74%) as compared with 194 (79%) for the repeat tissue culture (P greater than 0.1) cytotoxin (toxin B) test. Testing of 161 fresh stool samples found the latex test superior to tissue culture (P less than 0.05) in cases of CAD (90% positivity vs. 70%), with the two tests being equal in both non-CAD diarrheal and non-diarrheal control groups. In vitro evaluation of 61 C. difficile isolates found all (100%) to be producers of enterotoxin A, while only 53 (87%) produced toxin B. The latex test for C. difficile toxin detection is a rapid, simple test for use in the diagnosis in CAD.

Bacterial Toxins↗

MBCs for Staphylococcus aureus as determined by macrodilution and microdilution techniques.

MBC testing of clindamycin, methicillin, cephalothin, gentamicin, and vancomycin with 67 clinical isolates of Staphylococcus aureus was examined by both standard macrodilution tubes and commercial microdilution trays. Standard macrodilution failed to give reproducible (99.9% killing) MBC results, even when a strictly defined protocol was followed. Continuous shaking during incubation resulted in regrowth of more colonies than did stationary incubation. Vortexing of incubated tubes before subculture resulted in regrowth of more colonies than did careful transfer of the contents to sterile tubes before vortexing and subculture. No significant difference in MBCs was demonstrated by the use of log-phase versus stationary-phase inocula. Use of the multiprong inoculator for subculture from commercial microdilution trays was unsatisfactory because, although antibiotics evaluated were inactivated by subculture to a pH 5.5 agar plate coated with a beta-lactamase solution, the volume of broth transferred by the prongs was small and inconsistent, ranging from 0 to 3 microliter. Subcultures of commercial microdilution panels with a 1-microliter loop, 10-microliter pipette, and 100-microliter pipette were also evaluated. Results of MBC testing were most reproducible when the entire 100-microliter volume was aspirated from commercial microdilution wells after stirring and the contents of each well were spread over a separate sheep blood agar plate.

Anti-Bacterial Agents↗

Evaluation of the 24-h API 20A anaerobe system for identification of Clostridium difficile.

Accurate identification of Clostridium difficile is important when antibiotic-associated diarrhea or pseudomembranous colitis is suspected. Presumptive identification of C. difficile was made on the basis of microscopic features and colony characteristics on cycloserine, cefoxitin, fructose, and egg yolk agar medium. We studied the reliability of the 24-h API 20A anaerobe system for definitive identification of C. difficile. This system showed low dependability after the recommended 24 h of incubation by confirming the identity of only 54% of the isolates presumptively identified as C. difficile. There was a marked improvement in the system's capability after 48 h of incubation, when the identity of 95% of the isolates was confirmed.

Anaerobiosis↗

Evaluation of new blood culture processing systems.

The Antimicrobial Removal Device (ARD; Marion Scientific) was evaluated in vitro with simulated blood culture samples in fresh blood and clinically with samples from potentially septic patients to test its ability to remove antimicrobial agents and recover bacteria from blood culture specimens containing these drugs. In simulated specimens, the ARD was evaluated for adverse affects on microorganisms as well as compared with lysis-centrifugation (Isolator; Du Pont Co.), biphasic brain heart infusion bottles, and tryptic soy broth bottles for antimicrobial inactivation and organism recovery. There was no adverse effect of the ARD on organisms during a 4-h test period. The ARD was the only system to actually inactivate antimicrobial agents and removed greater than 99.2% of all antimicrobial agents tested from spiked and clinical specimens. Overall, with simulated blood culture specimens, the ARD recovered 90% of bacteria spiked into fresh blood containing antimicrobial agents, Isolator recovered 73%, biphasic brain heart infusion bottles recovered 31%, and tryptic soy broth bottles recovered 24%. In the clinical study, 43 of 86 clinically significant isolates were recovered only by ARD-assisted processing, 6 were recovered only by conventional processing, and 37 were recovered by both methods (the advantage of ARD processing over conventional processing in the clinical study was significant at P less than 0.001). Both clinical and simulated specimens demonstrated the ARD-associated blood culture processing to be the most efficient method for the isolation of microorganisms from specimens containing antimicrobial agents.

Anti-Bacterial Agents↗

Improved recovery of microorganisms from patients receiving antibiotics with the antimicrobial removal device.

Three hundred thirty-seven paired sets of blood cultures and ten sets of body fluid cultures from patients receiving antimicrobials were processed simultaneously by conventional methods with and without the use of an antimicrobial removal device (ARD) over a 13-month period of time in order to compare recovery rates. Forty-two significant pathogens were recovered: 19 by ARD processing only, four by conventional testing only, and the remaining 19 by both methods of processing. (P less than 0.001). Seventeen patient samples contained antimicrobics not active against the blood isolate (mainly Candida albicans). There was no difference (P greater than 0.1) between ARD-assisted and conventional methods in rate (frequency of cultures that were ultimately positive) or rapidity of detection of these microorganisms. In 17 subjects receiving antimicrobials active, in vitro, against the isolated microorganism, use of the ARD displayed a superior rate of recovery compared with conventional processing (P less than 0.001) and more rapidly detected organisms found both with and without ARD processing (P less than 0.04). Contamination rate was 3.7% for use of ARD compared with 0.6% without ARD (P less than 0.01). The ARD is a useful addition to conventional blood culture processing when used in a population of patients already receiving antimicrobial agents.

Adult↗

Prospective study of gram-stained stool smears in diagnosis of Clostridium difficile colitis.

Gram stains of stools from patients with diarrhea and control patients with no diarrhea were examined for a predominance of gram-positive rods and the presence of polymorphonuclear leukocytes. Results were compared with those from lower gastrointestinal endoscopy for pseudomembranes. Clostridium difficile culturing, and C. difficile toxin assay. The Gram stain was moderately difficult to interpret and was not useful in diagnosing diarrheal disease associated with C. difficile.

Clostridium Infections↗

Clinical laboratory evaluation of the thermonuclease test.

Simultaneous testing for clumping factor, coagulase, deoxyribonuclease, and thermonuclease was performed on 189 clinical isolates of gram-positive cocci with strong catalase activity to determine the suitability of the thermonuclease test as a routine procedure for the identification of Staphylococcus aureus. Positive reactions to all four tests were exhibited by 72 of the strains while 88 of the isolates gave uniformly negative results. Although discrepancies were found between the reactions of 29 organisms, differences were found between the reactions of 29 organisms, differences between tube coagulase ant thermonuclease results were rare. Greater than 90% of positive reactions for both tube coagulase and thermonuclease tests were detected within a four-hour incubation period. The thermonuclease test was found to be simple, reliable, inexpensive and rapid. This test gave easily interpretable reactions within an eight-hour workday, even when only one or two isolated colonies were used for testing. The thermonuclease test is well suited for use as a primary clinical laboratory procedure for the identification of Staphylococcus aureus.

Bacterial Proteins↗

Urine culture transport tubes: effect of sample volume on bacterial toxicity of the preservative.

Stable bacterial counts in urine specimens before culture are necessary to assure the accurate diagnosis of urinary tract infections. Preservative-containing tubes are commercially available for urine transport. As these tube containers are not always filled to the manufacturer's specifications, we studied the effects of stabilizer formation with low urine volumes. The Sage Urine Culture Tube and the Becton-Dickinson Urine Culture Kit were evaluated by using 30 cultures diluted in urine to 10(5) colony-forming units per ml. Both tube types were injected with 1, 2, 3, and 4 to 5 ml (tube capacity) of urine containing each culture. Specimens were held at 22 degrees C and cultured at 0, 4, and 24 h. Colony counts were corrected for the dilution due to the preservative. The Becton-Dickinson Urine Culture Kits were toxic to Escherichia coli and Klebsiella pneumoniae in specimens containing up to 2 ml of urine, and the minimum usable amount of urine for reliable results was 3 ml. The Sage Urine Culture Tube maintained the number of bacteria in 1 to 4.5 ml of urine in 83% of the specimens. However, the Sage tube was toxic to E. coli when held for 24 h. Quantitative counts of enterococci tended to significantly increase in specimens that contained 2 ml or more of urine with either system. The limitation of preservative-containing tubes for urine transport need to be recognized in order to avoid false-positive and false-negative results.

Bacteria↗

Concentrated gram stain smears prepared with a cytospin centrifuge.

A Cytospin slide centrifuge was used to concentrate 0.05- to 0.5-ml samples of cerebrospinal and other body fluids for Gram stain. Trials with cerebrospinal fluid containing known numbers of microorganisms indicated that the Cytospin increased the sensitivity of cerebrospinal fluid Gram stains by up to 2 logs compared with unconcentrated and conventional centrifuge smears. Cytospin-concentrated smears were prospectively compared with unconcentrated Gram-stained smears and bacteriological culture results for 80 clinical body fluid specimens. Bacteria were seen in unconcentrated smears of 9 of the 16 (56%) fluids which were infected, whereas Cytospin smears of 12 of the 16 (75%) showed bacteria. Cytospin smears revealed more bacteria and demonstrated better leukocyte morphology than did unconcentrated or conventionally centrifuged samples of small volumes of infected body fluids, allowing early diagnosis of infection.

Bacteriological Techniques↗

Activity of twenty-one antimicrobial agents including l-ofloxacin against quinolone-sensitive and -resistant, and methicillin-sensitive and -resistant Staphylococcus aureus.

There is a need to identify alternative agents to vancomycin for the treatment of infections with methicillin-resistant Staphylococcus aureus (MRSA). One candidate is the l isomer of ofloxacin (DR-3355). We tested 520 frozen MRSA isolates, 248 fresh MRSA isolates, and 375 fresh methicillin-susceptible S. aureus (MSSA) isolates from Minnesota, and 600 clinical isolates of S. aureus (150 MRSA and 450 MSSA) from Illinois. Over 90% of the MRSA strains were resistant to 32 micrograms/ml of oxacillin. Of the 520 frozen MRSA, 24% were susceptible to < or = 2 micrograms/ml ofloxacin, and an additional 74% were susceptible to ofloxacin between 8 and 16 micrograms/ml. More than 98% of all strains were susceptible to < or = 16 micrograms/ml ofloxacin or l-ofloxacin. All the quinolones had a bimodal distribution of in vitro activity, but for only ofloxacin and l-ofloxacin was activity confined to a very narrow range.

Anti-Bacterial Agents↗