Resistance of Candida species to fluconazole.
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Candida↗
Biomedical subjects
M A Pfaller
Publications and source records attributed to M A Pfaller.
Antifungal susceptibility testing of isolates from a randomized, multicenter trial of fluconazole versus amphotericin B as treatment of nonneutropenic patients with candidemia. NIAID Mycoses Study Group and the Candidemia Study Group.
The antifungal susceptibilities of 232 pathogenic blood stream Candida isolates collected during a recently completed trial comparing fluconazole (400 mg/day) with amphotericin B (0.5 mg/kg of body weight per day) as treatment for candidemia in the nonneutropenic patient were determined both by the National committee for Clinical Laboratory Standards M27-P macrobroth methodology and by a less cumbersome broth microdilution methodology. For amphotericin B, M27-P yielded a very narrow range of MICs (0.125 to 1 microgram/ml) and there were no susceptibility differences among species. For fluconazole, a broad range of MICs were seen (0.125 to > 64 micrograms/ml), with characteristic MICs seen for each species in the rank order Candida albicans < C. parapsilosis approximately equal to C. lusitaniae < C. glabrata approximately equal to C. krusei approximately equal to C. lipolytica. The MIC distribution for C. tropicalis was bimodal and could not be ranked. Both microdilution MICs were within one tube dilution of the M27-P MIC for > 90% of isolates with amphotericin B and for > or = 77% of isolates with fluconazole. For both methods, elevated MICs did not predict treatment failure. In the case of amphotericin B, the MIC range was too narrow to permit identification of resistant isolates. In the case of fluconazole, MICs for isolates associated with failure to clear the bloodstream consistently were equivalent to the median MIC for the given species. Successful courses of therapy were seen with four isolates from four patients despite MICs of > or = 32 micrograms/ml. As MICs obtained by M27-P and similar methods correlate with responsiveness to fluconazole therapy in animal models and in AIDS patients with oropharyngeal candidiasis, the lack of correlation in this setting suggests that the MICs for these isolates are at or below the relevant fluconazole breakpoint for this dose of fluconazole and patient setting and that host factors such as failure to exchange intravenous catheters were more important than MIC in predicting outcome.
Amphotericin B↗
Molecular characterization and multilaboratory evaluation of Enterococcus faecalis ATCC 51299 for quality control of screening tests for vancomycin and high-level aminoglycoside resistance in enterococci.
Studies were conducted to validate the use of Enterococcus faecalis ATCC 51299 (which is vancomycin resistant and resistant to high levels of gentamicin and streptomycin) and E. faecalis ATCC 29212 (which is susceptible to vancomycin and against which gentamicin or streptomycin and cell wall-active agents have synergistic kill activity) as controls in an agar screening test for vancomycin resistance and high-level streptomycin and gentamicin resistance and a broth microdilution screening test for high-level streptomycin and gentamicin resistance. Both organisms performed as expected in these tests and will serve as appropriate controls. However, E. faecalis ATCC 29212 was occasionally noted to produce light growth on the vancomycin screening plate with certain lots of agar. Quality control ranges for disk diffusion tests with disks with large amounts of streptomycin (300 micrograms) and gentamicin (120 micrograms) were established for E. faecalis ATCC 29212; zone limits are 16 to 22 mm for gentamicin and 14 to 19 mm for streptomycin. No zones for inhibition were seen when E. faecalis ATCC 51299 was tested with these high-content disks.
Aminoglycosides↗
Investigation of Candida albicans transmission in a surgical intensive care unit cluster by using genomic DNA typing methods.
An apparent outbreak of serious Candida albicans infections (n = 6) occurred in a surgical intensive care unit over a 4-week period. Four patients developed C. albicans bloodstream infections. An additional patient developed catheter-related C. albicans infection; the sixth patient developed an infection of cerebrospinal fluid. C. albicans was isolated from the hands of five health care workers (17%) and the throat of one health care worker (3%) during the outbreak investigation. Karyotyping and restriction endonuclease analysis of genomic DNA with BssHII of 23 C. albicans isolates from patients and the 6 health care worker isolates revealed 9 and 12 different patterns, respectively. Three of six patients appeared to be infected with the same C. albicans strain (two bloodstream infections and one cerebrospinal fluid infection). The hands of a health care worker were colonized with strain that appeared identical to an isolate from a patient prior to infection of the patient. However, restriction endonuclease analysis with SfiI found differences among the isolates determined to be identical by the other two methods. Karyotyping alone does not appear to be sufficient to differentiate between outbreak and control isolates. Restriction endonuclease analysis typing may be a more sensitive method than karyotyping alone in the investigation of a cluster of C. albicans infections. Furthermore, the use of more than one restriction enzyme may be necessary for optimal strain discrimination in restriction endonuclease analysis of genomic DNA.
Candida albicans↗
Comparison of visual and spectrophotometric methods of MIC endpoint determinations by using broth microdilution methods to test five antifungal agents, including the new triazole D0870.
A study to compare three different methods for reading MIC endpoints tested by the broth microdilution modification of the National Committee for Clinical Laboratory Standards (Villanova, Pa.) reference method was conducted. MICs of amphotericin B, flucytosine, fluconazole, itraconazole, and a new triazole, D0870, were determined for five reference yeast strains and 100 clinical isolates of Candida spp. MICs were read visually according to National Committee for Clinical Laboratory Standards guidelines from microdilution trays that had been (VS) and had not been (V) shaken. MICs were also determined spectrophotometrically (SP) at 492 nm. SP endpoints were determined as the concentrations resulting in a > or = 50% inhibition of growth (flucytosine and azoles) and a > or = 90% inhibition of growth (amphotericin B) relative to control growth. The five reference strains were tested nine times each against all five antifungal agents, and the MIC results for each reading method were compared with a 3-log2 dilution reference range determined by the macrodilution (M27-P) method. Overall, 84 to 100% of the MICs determined by V, 93 to 100% of those determined by VS, and 89 to 100% of those determined by SP fell within the 3-log2 dilution reference range for each reference strain and antifungal agent. Reproducibility was 99% for V and SP and 98% for VS. Agreement among the three methods of reading ranged from 97 to 99%. Excellent agreement among reading methods was also observed for all antifungal agents when tested against 100 clinical isolates. Agreement between the standard V method (no agitation) and VS ranged from 99 to 100%, and that between V and SP ranged from 89 to 99%. The VS and SP reading methods provided more definitive endpoints than the V method, which does not involve shaking.
Antifungal Agents↗
Quality control guidelines for National Committee for Clinical Laboratory Standards recommended broth macrodilution testing of amphotericin B, fluconazole, and flucytosine.
Amphotericin B, fluconazole, and flucytosine (5FC) were tested in a multilaboratory study to establish quality control (QC) guidelines for yeast antifungal susceptibility testing. Ten candidate QC strains were tested in accordance with National Committee for Clinical Laboratory Standards M27-P guidelines against the three antifungal agents in each of six laboratories. Each laboratory was assigned a unique lot of RPMI 1640 broth medium as well as a lot of RPMI 1640 common to all of the laboratories. The candidate QC strains were tested 20 times each against the three antifungal agents in both unique and common lots of RPMI 1640. A minimum of 220 MICs per drug per organism were generated during the study. Overall, 95% of the MICs of amphotericin B, fluconazole, and 5FC fell within the desired 3 log2-dilution range (mode +/- 1 log2 dilution). Excellent performance with all three drugs was observed for Candida parapsilosis ATCC 22019 and C. krusei ATCC 6258. With these strains, on-scale 3 log2-dilution ranges encompassing 96 to 99% of the MICs of all three drugs were established. These two strains are recommended for QC testing of amphotericin B, fluconazole, and 5FC. Reference ranges were also established for an additional four strains for use in method development and for training. Four strains failed to perform adequately for recommendation as either QC or reference strains.
Amphotericin B↗
Comparison of four DNA-based methods for strain delineation of Candida lusitaniae.
Four methods for the accurate delineation of epidemiologically related and unrelated strains of Candida lusitaniae were compared. Three pulsed-field electrophoretic methods, including two contour-clamped homogeneous field gel electrophoresis methods (EKP-1 and EKP-2) yielding electrophoretic karyotype patterns of intact chromosomal DNA and a method in which the chromosomal DNA was macrodigested with the endonuclease SfiI prior to pulsed-field electrophoresis (MDP), and a random amplified polymorphic DNA (RAPD) assay were evaluated. A selected panel of 21 well-characterized isolated representing 13 strains of C. lusitaniae, including 7 epidemiologically related isolates of one strain (group I-A), 3 epidemiologically related isolates of another strain (group I-B), and 11 epidemiologically unrelated isolates (group II), were tested. All isolates were coded and tested in a blinded manner. All seven group I-A isolates were confirmed to be a single strain by the EKP-1 and MDP methods, and the three group I-B isolates were shown to be a single strain by the EKP-1, EKP-2, MDP, and RAPD methods. Subtle differences were noted with two of the group I-A isolates by the EKP-2 method, whereas three of these isolates were different by the RAPD method. Each group II isolate had distinct patterns by all four methods. These data support the fact that the three pulsed-field electrophoretic methods and the RAPD method can be used to delineate strains of C. lusitaniae. The EKP-1, EKP-2, and MDP gave results that correlated with the epidemiologic characteristics of the isolates tested in the study, whereas the RAPD method was perhaps too sensitive in detecting DNA changes for epidemiologic studies.
Candida↗
The use of molecular techniques in the epidemiology and control of infectious diseases.
The ability to identify specific strains within a given species of pathogen is an important aid in the rational development of effective measures to prevent and control nosocomial infections. The efforts of both microbiologist and hospital epidemiologic are facilitated greatly by the availability of the newer molecular epidemiologic typing techniques. Although these methods clearly have limitations, they generally are a significant improvement over the more conventional typing methods, many of which are too cumbersome, insensitive, and time-consuming to be of practical value for epidemiologic evaluations. Based on current experience, the molecular typing methods that appear to be the most practical and useful for both large and small scale epidemiologic studies are the DNA-based methods of REAP and PFGE. Many questions remain concerning the appropriate role of molecular typing methods in the clinical microbiology laboratory. Often, molecular typing may be performed more efficiently in a reference laboratory. In contrast, selected methods such as plasmid analysis are well within the scope of clinical microbiology laboratories and may be an important adjunct to the hospital infection control effort. Given the limitations of the available methods and the complex nature of patients at risk for nosocomial infections, it is imperative that these methods be employed with clear epidemiologic objectives in mind. Typing should always include unrelated as well as epidemiologically related isolates and, whenever possible, all organisms should be typed under identical conditions, preferably within the same test run. In addition, results are most effectively used to supplement rather than to replace hypotheses and questions thoughtfully developed by the clinician or epidemiologist. Ideally, typing is performed independently by the laboratory to avoid bias, but the results are applied collaboratively to ensure that both the potential insights and the unavoidable ambiguities presented by the results are clearly appreciated. Additional studies based upon sound epidemiologic principles will help clarify the role of the various molecular typing methods as epidemiologic markers and advance our understanding of the epidemiology of nosocomial infections.
Bacterial Infections↗
Epidemiology of candidiasis.
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Antifungal Agents↗
A swimming-associated outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7 and Shigella sonnei.
BACKGROUND: In the summer of 1991, simultaneous outbreaks of bloody diarrhea and hemolytic-uremic syndrome caused by Escherichia coli O157:H7 and of bloody diarrhea caused by Shigella sonnei were traced to a lakeside park near Portland, Oregon. METHODS: We identified cases primarily from routine surveillance reports. In case-control studies, the activities of persons with park-associated E. coli O157:H7 or S. sonnei infections were compared independently with those of three sets of controls. We also evaluated environmental conditions at the park and subtyped the bacterial isolates. RESULTS: We identified 21 persons with park-associated E. coli O157:H7 infections (all of them children; median age, six years) and 38 persons with S. sonnei infections (most of them children). These 59 people had visited the park over a 24-day period. Their illnesses were not associated with food or beverage consumption. All the case patients reported swimming, however, and in case-control studies swimming was strongly associated with both types of infection (P = 0.015 or less). The case patients were more likely than the controls to report having swallowed lake water, and they had spent more time in the lake. Numbers of enterococci indicative of substantial fecal contamination (geometric mean, > 50 per deciliter) were detected in the swimming area during some but not all of the outbreak period. Park-associated E. coli O157:H7 isolates were identical by pulsed-field gel electrophoresis and were distinguishable from other isolates in the Portland area. CONCLUSIONS: Lake water that was fecally contaminated by bathers was the most likely vehicle for the transmission of both the E. coli O157:H7 and the S. sonnei infections. The unusually prolonged outbreak suggests both the survival of these enteric organisms in lake water and a low infectious dose.
Case-Control Studies↗
Long-term efficacy of intranasal mupirocin ointment. A prospective cohort study of Staphylococcus aureus carriage.
BACKGROUND: We investigated the long-term effect of a single 5-day application of intranasal mupirocin calcium ointment on Staphylococcus aureus nasal and hand colonization. The subjects were 68 healthy volunteers who were health care workers with stable S aureus nasal carriage and who had participated in a randomized, double-blind placebo-controlled clinical trial of intranasal mupirocin ointment. METHODS: A 1-year prospective cohort study of S aureus nasal carriers after treatment with active drug or placebo was performed. Cultures were obtained from all subjects 6 and 12 months after therapy. All subjects returned for the 6-month visit; 63 (93%) were examined at 1 year. The major outcome measure was the relative proportion of any S aureus cultured at either site at 6 and 12 months. The S aureus isolates were typed by restriction endonuclease analysis of plasmid DNA and by antibiotic susceptibility tests; the similarity of nasal and hand isolate "fingerprints" was compared. RESULTS: At 6 months, nasal carriage was 48% in the treatment group vs 72% in controls (relative risk, 0.68; 95% confidence interval, 0.45 to 1.02; P = .054); at 1 year, nasal carriage was 53% vs 76%, respectively (relative risk, 0.70; 95% confidence interval, 0.48 to 1.02; P = .056). Hand carriage at 6 months was significantly reduced among mupirocin recipients relative to controls (15% and 48%; P = .04, adjusted for the baseline rate of hand carriage). Thirty-six percent of treated subjects were recolonized in the nares with a new strain at 1 year, whereas 34% had reisolation of the original strain after initially negative posttherapy cultures. During the year of follow-up, hand carriage was observed at least once in two thirds of the subjects. Nearly all of the hand isolates (87%) exactly matched the subjects' coincident nasal plasmid fingerprint and antibiogram type. CONCLUSIONS: A single brief treatment course of intranasal mupirocin was effective in reducing nasal S aureus carriage for up to 1 year. When S aureus was recovered after nasal decolonization, the new isolate was as likely to represent colonization with a new strain as reisolation of the original strain. Staphylococcus aureus hand carriage was significantly decreased 6 months after therapy, further implicating the nares as the primary reservoir site for hand carriage.
Administration, Intranasal↗
Molecular epidemiology for local outbreaks of methicillin resistant Staphylococcus aureus (MRSA). The need for several methods.
Subtyping isolates may be useful for epidemiological studies of methicillin-resistant-Staphylococcus aureus (MRSA) outbreaks. Among subtyping methods, DNA-based techniques have been applied very effectively for this purpose. An outbreak of MRSA infections took place in one hospital in Barcelona early during 1991. From the beginning of the outbreak to December 92, 70 MRSA isolates from different patients and sources were collected. All strains were evaluated by restriction endonuclease analysis of plasmid DNA (REAP) and macrorestriction endonuclease analysis of genomic DNA using Sma I and pulsed-field-gel-electrophoresis (PFGE). Plasmid screening and REAP using Hind III demonstrated two plasmid subtypes: subtype A showing a large plasmid, and subtype B showing the same large plasmid plus a smaller one. Subtypes A and B corresponded to the more recent and older isolates, respectively, suggesting the loss of the small plasmid during the epidemic. PFGE using Sma I displayed two closely related profiles (PFGE subtype A and A'; CS = 0.90). These subtypes were different from those subtypes exhibited from 4 methicillin-susceptible-Staphylococcus aureus (MSSA) isolates from the same hospital and from 2 epidemiologically unrelated MRSA isolates. Almost all isolates showing PFGE subtype A preceded those isolates showing PFGE subtype A'. This fact and the similarity between both subtypes suggested minor chromosomal DNA rearrangement during the outbreak from a unique strain. While PFGE using Sma I is a useful tool in evaluation of clonal dissemination, our data suggest epidemic or local outbreaks may need several methods to best delineate the source and spread of MRSA strains. The reproducibility and discriminatory power of REAP makes it a useful adjunct in this context.
Anti-Bacterial Agents↗
Quality control parameters for broth microdilution susceptibility testing of amoxicillin and amoxicillin-clavulanic acid.
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Amoxicillin↗
Revision of quality control limits for broth microdilution tests of fleroxacin and gentamicin with Pseudomonas aeruginosa ATCC 27853.
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Escherichia coli↗
Prevalence of important pathogens and the antimicrobial activity of parenteral drugs at numerous medical centers in the United States. II. Study of the intra- and interlaboratory dissemination of extended-spectrum beta-lactamase-producing Enterobacteriaceae.
The incidence of extended-spectrum beta-lactamases (ESBL)-producing Klebsiella pneumoniae and Escherichia coli has been increasing rapidly, and they are probably even more prevalent than is currently recognized because of difficulties in their detection by the clinical microbiology laboratory. In addition, several outbreaks associated with these multiresistant strains have been reported. In the present study we evaluated 30 clinical isolates (27 K. pneumoniae from 11 hospitals and three E. coli from three hospitals) that were resistant or intermediately susceptible to ceftazidime and/or cefuroxime. The main objective of the study was to evaluate the intra- and interhospital dissemination of ESBL-producing strains. The isolates were tested for susceptibility to ceftazidime, cefuroxime, gentamicin, and ofloxacin, and the ability of various susceptibility testing methods to detect resistance to these beta-lactams was evaluated. The production of ESBL was assessed by the disk approximation synergy test, and typing was performed by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA. ESBL production was demonstrated in 15 K. pneumoniae (from seven hospitals) and in one E. coli strain. Most ESBL-producing isolates demonstrated cross resistance with gentamicin and ofloxacin. Chromosomal DNA analysis by PFGE exhibited a great genomic variability among ESBL-producing isolates. Our results also indicated the occurrence of both intra- and interhospital dissemination of these multiresistant strains.
Bacteriological Techniques↗
Molecular epidemiology of gastric colonization by Enterococcus faecalis in a surgical intensive care unit.
We applied restriction endonuclease analysis of genomic DNA using pulsed field gel electrophoresis (PFGE) to study gastric colonization with Enterococcus faecalis among patients hospitalized in the surgical intensive care unit (SICU). Isolates were obtained by culturing prospectively the gastric contents of 140 patients in the SICU. In addition, cultures of respiratory specimens were obtained daily and cultures of blood, normally sterile body fluids, wounds, and urine were obtained when indicated clinically. A total of 177 isolates were obtained from 45 patients. Concentrations of E. faecalis in gastric fluid ranged from 1 x 10(2) colony forming units (CFU)/ml to greater than 5 x 10(7) CFU/ml (mean 8.0 x 10(6) CFU/ml). Overall, 33 different DNA types were identified by PEGE. In examining strain variation among isolates obtained from multiple anatomic sites over time, we found that the same DNA type was recovered from gastric aspirates, sputum, and wounds in a given patient and that these strains were carried over time. In general, given individuals were colonized with their own unique DNA type; however, one DNA type (type C) was shared by 11 different patients, and seven DNA types were shared by two individuals each. These results demonstrate the potential importance of gastric colonization as a reservoir for nosocomial strains of E. faecalis in an SICU setting.
Colony Count, Microbial↗
Multicenter comparison of a colorimetric microdilution broth method with the reference macrodilution method for in vitro susceptibility testing of yeast isolates.
This multicenter study was performed to compare a colorimetric microdilution method with the NCCLS M27-P reference macrodilution method for the testing of yeast isolates against amphotericin B, fluconazole, and 5-fluorocytosine (5FC). Testing was performed on ten yeast isolates in five independent laboratories. All sites tested each isolate a total of 20 times with both methods. MICs were read after 48 h incubation. The macrodilution MIC reference range was defined as the modal MIC +/- 1-log2 dilution for each organism-antifungal agent combination. Agreement between the M27-P reference range results and the microdilution MICs was 86% with amphotericin B, 90% with fluconazole, and 93% with 5FC. Based on these data, it is apparent that new approaches, such as the colorimetric microdilution method, will provide MIC values comparable to the M27-P macrodilution method in a format that is more practical for use in a busy clinical laboratory.
Amphotericin B↗
Comparison of identification systems for Staphylococcus epidermidis and other coagulase-negative Staphylococcus species.
Three commercially available systems (API Staph-Trac, API 20GP, and Vitek GPI), used to identify coagulase-negative staphylococci, were evaluated against 277 bloodstream isolates, including 94 isolates of Staphylococcus epidermidis and 183 isolates of other coagulase-negative Staphylococcus species. The conventional method of Kloos and Schleifer served as the reference method. Controls included 14 ATCC type culture strains of coagulase-negative staphylococci. The API Staph-Trac system showed the highest rate of agreement with reference method, correctly identifying 73% of the isolates. The Vitek GPI System had an overall rate of agreement of 67% and the API 20GP system correctly identified 61%. The API Staph-Trac system correctly identified 94% of the isolates of S. epidermidis compared with 64% by both Vitek GPI and API 20GP. The most common error for both Vitek GPI and API 20GP systems was the failure to identify organisms contained within the database of the systems. Because none of the tested commercial identification systems identified "non-epidermidis" coagulase-negative Staphylococcus species with a high degree of accuracy, the systems need to be markedly improved or new systems developed.
Bacteremia↗