Biomedical subjects
Publications and source records attributed to B S Reisner.
The performance of Vitek cards GPS105 with software version VTK-R07.01 for detection of oxacillin resistance in coagulase-negative staphylococci (CoNS) was compared to disk diffusion and PCR detection for mecA. The sensitivity and specificity of the Vitek GPS105 method were 97.6 and 85.5%, respectively.
Bafilomycin A(1), a selective inhibitor of V-type H(+)-translocating ATPase (V-ATPase), may be a useful adjunct in cancer chemotherapy (Altan et al. [1998] J Exp Med 187:1583-1598). Therapeutic uses of the enzyme inhibitor need to consider the agent's potential effects on normal (nontumor) cells. This study determined the effects of bafilomycin A(1) on resident alveolar macrophages (mphi). Treatment of alveolar mphi with bafilomycin A(1) (10 microM, 1 h) caused a significant decrement in cytosolic pH. This was accompanied by marked alteration of mphi bactericidal capabilities. The enzyme inhibitor caused a marginal reduction in the phagocytosis of opsonized Staphylococcus aureus and significantly suppressed intracellular killing of the phagocytosed bacteria. In keeping with the effects on intracellular killing, bafilomycin A(1) significantly reduced the production of reactive oxygen species (ROS). On the other hand, cell spreading was enhanced significantly by bafilomycin A(1). Comparable changes in ROS generation and mphi spreading were produced by altering cytosolic pH through changes in extracellular pH (pH(o)) in the absence of bafilomycin A(1). These findings suggest that the agent's effects on ROS production and mphi spreading were related to the accompanying changes in cytosolic pH. The enzyme inhibitor also altered mphi morphology, leading to the shortening of microvilli and focal loss of surface ruffles. These morphologic effects differed from those produced by altering cytosolic pH by changes in pH(o). The results demonstrate that V-ATPase activity is an important determinant of mphi functioning and structure. Therapeutic use of V-ATPase inhibitors might be expected to compromise the bactericidal activity of alveolar mphi.
Four methods (bile solubility, optochin, latex agglutination, and DNA probe) were compared for identification of clinical isolates of Streptococcus pneumoniae. Of 209 isolates tested, 151 (Group I) were selected based on typical colony morphology of S. pneumoniae, while 58 (Group II) were selected on the basis of alpha-hemolysis alone. Using the DNA probe as a reference method, 141 isolates from Group I and 10 from Group II were identified as S. pneumoniae. The optochin disk test and the latex agglutination test both exhibited a 100% sensitivity and specificity for Group I isolates; bile solubility identified all but 1 isolate in this group. For Group II isolates, the sensitivity and specificity of optochin testing was 100%, 80 and 94% for latex and 80 and 100% for bile. The results of this study indicate that all methods tested give reliable results for isolates with typical colony morphology of S. pneumoniae. Bile solubility and latex may not be as reliable when testing alpha-hemolytic colonies without colony morphology typical S. pneumoniae.
Oxacillin resistance in Staphylococcus aureus is mediated by the mecA gene, resulting in production of penicillin-binding protein 2a (PBP2a), which is not present in the oxacillin susceptible strains. We evaluated the ability of a 30-min latex agglutination (LA) test (Seiken, Tokyo, Japan) to detect production of PBP2a in 315 clinical isolates of S. aureus. The LA results were compared with results of susceptibility testing using the Vitek GPS-SV test card. The latex test was positive for all 206 isolates determined to be methicillin resistant by Vitek (sensitivity 100%), the latex test was negative for 108 of 109 isolates determined to be oxacillin susceptible by Vitek, and the latex test was positive for 1 isolate determined to be susceptible by Vitek (specificity 99.1%). The discrepant isolate was negative for the mecA gene by polymerase chain reaction (PCR). The LA test is a rapid and reliable method for detecting oxacillin resistant S. aureus.
We compared a new assay for Toxoplasma IgM on the Access analyzer (Beckman Coulter, Inc., Chaska, MN, USA), a random access instrument based on the principle of paramagnetic particle enzyme immunoassay with an enzyme-linked immunosorbent assay (ELISA) (Zeus Scientific, Inc., Raritan, NJ, USA) and an immunofluorescent assay (IFA) (Gull Laboratories, Inc., Salt Lake City, UT, USA). Four hundred fresh, unfrozen clinical samples from pregnant women (n = 154), HIV positive patients (n = 41), and patients in whom infection with Toxoplasma gondii was suspected (n = 200) were collected and assayed over a three month period. The specificity of the Access assay was compared to the consensus results. Results that were discrepant between the ELISA and IFA were resolved using a third IFA (Zeus). Once resolved, the specificity for the Access assay, the Zeus ELISA and the Gull IFA were 99.22%, 97.91%, and 99.45%, respectively. We conclude that the Access assay specificity is comparable to consensus results, minimizing false positive results; and because it is a random access instrument, it may be preferable over batch methods.
The performance of the AccuProbe Group B Streptococcus Culture Identification Test (Gen-Probe Incorporated, San Diego, CA, USA) for the detection of group B streptococci (GBS) directly from LIM broth cultures of vaginal-anorectal swab specimens from pregnant women (two swabs per patient in most cases) was evaluated by comparing results to those of conventional GBS culture. Of 411 specimens analyzed, 82 were positive and 312 were negative for GBS by both methods. After initial testing, the percent agreement was 95.9%. The initial sensitivity, specificity, and positive and negative predictive values for the AccuProbe test were 90.1%, 97.5%, 91.1%, and 97.2%, respectively. Results were discrepant for 17 specimens: eight were GBS positive by probe and negative by culture; nine were negative by probe and positive by culture. To resolve discrepancies, culture plates were re-examined for GBS colonies, AccuProbe testing was repeated on the initial LIM broth cultures, and the second swab (if received) was inoculated to LIM broth for AccuProbe testing after overnight incubation. After discrepant resolution testing, the percent agreement between the two test methods was 97.8%. The final sensitivity, specificity, and positive and negative predictive values for the AccuProbe test were 95.6%, 98.4%, 94.6%, and 98.7%, respectively. These data suggest that the AccuProbe test is a reliable method for detecting GBS in vaginal-anorectal specimens, providing results more rapidly than conventional culture. However, strict adherence to the manufacturer's test protocol is necessary to limit technical errors.
The increasing prevalence of drug-resistant bacteria is attributed to the extensive use of antibiotics, which causes selective pressure on the nasopharyngeal bacterial flora. Shortened courses of antibiotics have been proposed to decrease the development of resistant strains. We determined the effect of a single intramuscular dose of ceftriaxone (50 mg/kg) on the nasopharyngeal bacterial flora in 167 children (median age 13 mo) with acute otitis media. Nasopharyngeal samples for bacterial culture were obtained before and 5 d after treatment with ceftriaxone. Before treatment, Moraxella catarrhalis was isolated in 99 (59%) children, Streptococcus pneumoniae in 87 (52%), and Haemophilus influenzae in 53 (32%). After treatment, M. catarrhalis was found in 62 (37%) children, which constitutes a 37% decrease in the colonization rate by this pathogen (p < 0.001). S. pneumoniae was isolated in 50 (30%; 43% decrease) and H. influenzae in 17 (10%; 68% decrease) children after treatment (p < 0.001 for both). Before treatment, 60% of pneumococcal isolates were sensitive to penicillin, 26% were of intermediate susceptibility, and 14% were penicillin-resistant. Eradication of S. pneumoniae occurred mainly in children with penicillin-sensitive isolates. As a consequence, only 24% of pneumococcal isolates that remained after treatment were sensitive to penicillin, 59% were penicillin-intermediate, and 16% were penicillin-resistant. A single dose of ceftriaxone resulted in significant changes in the nasopharyngeal bacterial flora, increasing the relative prevalence of pneumococcal strains with decreased susceptibility to penicillin.
OBJECTIVE: To establish an efficient and sensitive technique for recovering vancomycin-resistant enterococci (VRE) from perianal and environmental samples collected during implementation of control measures for an outbreak of VRE. DESIGN: Perianal and environmental samples were collected in triplicate on sterile swabs. One swab was used to inoculate a selective broth medium containing 6 pg of vancomycin and 8 pg of ciprofloxacin per mL, one to inoculate Campylobacter agar containing 10 microg/mL of vancomycin, and one to inoculate Enterococcosel agar containing 8 microg/mL of vancomycin. SETTING: Samples were collected in the intensive care units of a 600-bed university hospital over a period of 2 months. SAMPLE SELECTION: Patients and their immediate environment were sampled if they resided in a ward with a patient known to be colonized or infected with VRE. RESULTS: Of the 88 perianal samples obtained from 63 patients, 37 were positive for VRE by broth culture, with 36 also recovered on both types of solid media (sensitivity, 97.3%; negative predictive value, 98.1%). Of the initial samples collected from each of the 63 patients, 20 were positive for VRE by all methods. Of the 500 environmental samples cultured, 139 were positive for VRE in broth, with only 33 recovered on Campylobacter agar (sensitivity, 23.7%; negative predictive value, 77.2%) and 22 on Enterococcosel agar (sensitivity, 15.8%; negative predictive value, 75.2%). CONCLUSIONS: Our data indicate that, when performing surveillance cultures during an outbreak of VRE, use of an enrichment broth medium is required to recover VRE contaminating environmental surfaces; however, direct inoculation to selective solid medium is adequate to recover VRE in patient perianal specimens.
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The standard method for detecting antinuclear antibodies (ANAs) is by immunofluorescence assay (IFA), a method that is labor intensive and subjective. In an attempt to overcome these limitations, several commercial enzyme immunoassays (EIAs) have been developed. We report the results of our evaluation of the ANA Microplate EIA (Sanofi Diagnostics Pasteur, Chaska, MN). For the evaluation, 808 serum samples were tested by EIA and IFA; 52 specimens were positive by both assays, 561 were negative by both assays, 91 were positive by EIA only, and 3 were positive by IFA only. Borderline results (not positive or negative) were obtained for 101 specimens, which were excluded when calculating the sensitivity, specificity, and positive and negative predictive values of this assay, which were 94.6%, 86.0%, 36.4%, and 99.5%, respectively. Because of its high negative predictive value, this assay can be used reliably to detect ANA-negative samples; however, the low positive predictive value indicates that EIA-positive specimens should be retested by an IFA to determine the final result.
In this study, disk diffusion testing with ceftizoxime and cefuroxime was evaluated for use in predicting the susceptibility of Streptococcus pneumoniae to ceftriaxone and cefotaxime. Of the 194 isolates included in this study, 138 were susceptible, 34 were intermediate, and 22 were resistant to cefotaxime by MIC testing; 138 isolates were susceptible, 35 were intermediate, and 21 were resistant to ceftriaxone by MIC testing. A zone of inhibition around the cefuroxime disk of >/=32 mm correctly categorized 101 of 138 isolates as susceptible to cefotaxime and ceftriaxone. A zone of inhibition around the ceftizoxime disk of >/=26 mm correctly categorized 111 of 138 isolates as susceptible to cefotaxime and 114 of 138 as susceptible to ceftriaxone. We conclude that disk diffusion can separate S. pneumoniae isolates susceptible to ceftriaxone and cefotaxime from those that are not susceptible. Isolates not falling into the susceptible category by disk diffusion require additional testing to determine the MIC.
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.
OBJECTIVE: To compare the ability of the Vitek GPS-TB card with disk diffusion testing for determining the susceptibility of enterococci to vancomycin. DESIGN: Vitek susceptibility testing was performed using the GPS-TB card and software version R05.03. Disk diffusion susceptibility testing was performed according to National Committee for Clinical Laboratory Standards guidelines. When discrepancies occurred between the interpretation of Vitek and disk diffusion, both tests were repeated and the epsilometer test (E test) and agar screen containing 6 microgram/mL vancomycin were performed. RESULTS: Of 415 isolates tested, 313 were susceptible to vancomycin and 97 were resistant to vancomycin by both test methods. Two isolates were intermediate by Vitek and resistant by disk diffusion, 2 were intermediate by Vitek and susceptible by disk diffusion, and 1 was susceptible by Vitek and intermediate by disk diffusion. All but 1 of these latter 5 isolates (intermediate by Vitek and susceptible by disk diffusion) were available for retesting. On repeat testing, the 2 isolates that were intermediate by Vitek and resistant by disk diffusion were resistant by both methods, the 1 isolate that was intermediate by Vitek and susceptible by disk diffusion was susceptible by both methods, and the isolate that was susceptible by Vitek and intermediate by disk diffusion was also susceptible by both methods. These results were confirmed by E test and agar screen. CONCLUSION: We found the results of the GPS-TB card compared well with disk diffusion. However, isolates with intermediate results by Vitek should be retested using another method, such as the E test.
The CPS ID 2 (CPS) chromogenic agar was compared to routine media for use in the isolation, enumeration, identification, and susceptibility testing of bacteria recovered from urine specimens. Of 487 urine specimens, 318 were culture negative, 12 were positive on CPS only, 16 positive on routine only, and 141 positive on both. The enumeration of microorganisms agreed for 96 of the 141 cultures. Fewer organisms were recovered on CPS for 25 cultures, more for 20 cultures. The identification of bacteria from CPS and routine media agreed for all isolates. Susceptibility testing was performed for 100 isolates. The categorical susceptibility test results from isolates grown on CPS agreed with results from routine media for 77 isolates. For the remaining 23 isolates, one or more discrepancies were seen involving 16 different antimicrobial agents; 27 minor, 1 major, and 6 very major errors. After additional testing, there were 10 confirmed errors, 7 minor and 3 very major errors. This study demonstrates that CPS agar is a reliable medium for culturing urine. Susceptibility testing directly from this medium resulted in low error rates for all antimicrobial agents tested.
The Captia Syphilis IgG enzyme immunoassay (EIA) was evaluated for use in conjunction with the rapid plasma reagin test (RPR) as a method to test for syphilis. A total of 1,288 serum specimens were tested by the routine laboratory protocol of the RPR followed by microhemagluttination assay for Treponema pallidum (MHA-TP) testing of RPR-reactive sera as well as the EIA-RPR protocol in which the automated EIA followed by a manual RPR test for EIA-positive specimens is used. When using the routine protocol, 131 specimens were initially reactive by the RPR, and 113 of these were reactive by MHA-TP. When using the EIA-RPR protocol, 170 specimens were initially positive by EIA, and of these, 112 were RPR reactive, indicating active disease. When compared to the routine protocol, the EIA-RPR protocol had sensitivity, specificity, and positive and negative predictive values of 96.5, 99.7, 97.3, and 99.7%, respectively. After resolution of discrepancies by additional testing, the adjusted sensitivity, specificity, and positive and negative predictive values were 100, 99.8, 98.3, and 100%, respectively. This evaluation demonstrates that when used in conjunction with the RPR, the Captia Syphilis EIA is a reliable method by which to test for syphilis.
SETTING: Mycobacterium avium complex (MAC) isolates grown in the presence of clarithromycin, ciprofloxacin, ethambutol or rifabutin were examined by electron microscopy for drug-induced ultrastructural changes. OBJECTIVE: To further the understanding of the activity of clarithromycin, ciprofloxacin, ethambutol and rifabutin against MAC. DESIGN: Four MAC isolates, 1 control and 3 patient, were cultured for 24 or 72 hours in liquid medium containing one of two concentrations of an antimicrobial agent prior to examination by electron microscopy. RESULTS: One or more of the following ultrastructural changes was observed after exposure to the antimicrobial agent: disorganized nucleoid and cytoplasm, condensation of nucleoid, vacuolization, enlargement of the periplasmic space with or without vesicles, intracytoplasmic lipid-like inclusions, and cell lysis. Changes were seen after as little as 24 hours of exposure to the antimicrobial agent and were more evident with the higher of the two concentrations of the drug. Changes were present regardless of the minimum inhibitory concentration (MIC) of the antimicrobial agent against the isolates tested, but were more pronounced if the MIC was less than the concentration of drug tested. CONCLUSION: Consistent ultrastructural changes were observed following exposure to a given antimicrobial agent. No drug-specific changes were identified.
The reliability of mycobacteria growth indicator tubes (MGIT) for determining the susceptibility of Mycobacterium tuberculosis to isoniazid (0.1 microgram/ml) and rifampin (2.0 micrograms/ml) was evaluated by comparing MGIT results to those obtained by the radiometric BACTEC TB method. We tested 29 isolates, many selected for resistance. The turnaround times were 3-8 days (median 6) for MGIT and 4-10 days (median 6) for BACTEC. Isoniazid results by both methods agreed for all isolates tested: 20 resistant and nine susceptible. Rifampin results agreed for 28 isolates: 10 resistant and 18 susceptible. One isolate yielded discrepant results: resistant to rifampin by BACTEC, but susceptible by MGIT. This isolate was also rifampin-resistant by the traditional Method of Proportion and when tested by the MGIT method using 1.0 micrograms/ml of rifampin. The MGIT system is a nonradiometric alternative to the BACTEC for rapid susceptibility testing of M. tuberculosis to isoniazid and rifampin; however, additional testing is needed to determine the optimal concentration of rifampin.
To evaluate the utility of Gen-Probe AccuProbes for the identification of mycobacteria directly from BACTEC TB 12B vials containing acid-fast bacilli, culture results for 11,375 clinical specimens other than blood received from 1 January 1992 to 30 September 1993 were reviewed retrospectively. During this period, a total of 359 of 11,375 BACTEC vials were positive for acid-fast bacilli and were evaluated for mycobacteria with one or more probes: 224 were probed for Mycobacterium tuberculosis complex, 253 were probed for Mycobacterium avium complex, 64 were probed for Mycobacterium kansasii, and 77 were probed for Mycobacterium gordonae. After initial testing with the probes, 75 vials were positive for M. tuberculosis complex, 99 were positive for M. avium complex, 11 were positive for M. kansasii, and 55 were positive for M. gordonae. Repeat testing of vials that were initially probe negative or testing of colonies from subcultures of these vials identified an additional 11 M. tuberculosis, 27 M. avium complex, 1 M. kansasii, and 9 M. gordonae that were not detected on initial screening. On the basis of these data, the percentage of organisms identified directly from the BACTEC TB 12B vials upon initial screening with each of the four AccuProbes was 87.2% for M. tuberculosis complex, 78.6% for M. avium complex, 91.7% for M. kansasii, and 85.9% for M. gordonae.