Colorimetric method for susceptibility testing of voriconazole and other triazoles against Candida species.

A microdilution assay using Alamar Blue, a colorimetric indicator, was compared with the NCCLS macrodilution broth assay for voriconazole, fluconazole, and itraconazole against Candida albicans, Candida glabrata, and Candida krusei. Concordance (+/- 2 dilutions) between the two methods was highest for voriconazole (98.3%), and for fluconazole and itraconazole it was 94.3 and 95.4%, respectively. Twenty-six of 32 (81.2%) discordant readings (> or = 3 dilutions different) were noted in C. glabrata isolates, and all but two isolates showing discordance had higher minimum inhibitory concentration readings with the colorimetric method.

In vitro activity of voriconazole against Candida species.

The in vitro activity of voriconazole was compared with that of itraconazole and fluconazole against 181 isolates of Candida albicans, 124 isolates of Candida glabrata, and 20 isolates of Candida krusei obtained from the early 1980s through the mid-1990s. Voriconazole had greater intrinsic activity than fluconazole or itraconazole against all three Candida species. For C. glabrata, C. krusei, and C. albicans, the MIC50 values for voriconazole were 1 microgram/mL, 0.5 microgram/mL, and 0.01 microgram/mL, respectively compared with fluconazole MIC50 values of 8 micrograms/mL, 64 micrograms/mL, and 0.25 microgram/mL, respectively. If isolates from AIDS patients were excluded, MIC values for isolates from the 1990s were no higher than those noted for isolates from the 1980s. Voriconazole, a new triazole antifungal agent, appears to have enhanced activity against these three species of Candida; the clinical relevance of these findings should be studied in treatment trials.

Susceptibility of ciprofloxacin-resistant staphylococci and enterococci to trovafloxacin.

The susceptibilities of clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and enterococci obtained over a 13-year period were tested for trovafloxacin, a new fluoroquinolone, and ciprofloxacin. For MRSA, MIC50 values for trovafloxacin increased from 0.03 microgram/ml to 1 microgram/ml from 1984-1985 to 1995-1996, but were lower than those noted for ciprofloxacin, which increased from 0.25 microgram/ml to > 8 micrograms/ml during the same period. Trovafloxacin also showed lower MIC50 values (0.12-0.25 microgram/ml) than ciprofloxacin (MIC50 of 0.5-1 microgram/ml) for E. faecalis isolates from 1985 through 1991, but against E. faecium and E. faecalis isolated in 1995-1996, the MIC50 values for both trovafloxacin and ciprofloxacin were 2 micrograms/ml and the MIC90 values were > or 8 micrograms/ml. Thus, cross-resistance between fluoroquinolones was shown for both enterococci and MRSA. Whether the greater intrinsic activity of trovafloxacin might allow its use in treating MRSA and enterococcal infections remains to be seen.

Saccharomyces cerevisiae infections and antifungal susceptibility studies by colorimetric and broth macrodilution methods.

Saccharomyces cerevisiae was isolated in large numbers from operative specimens from two patients with perforated bowel and peritonitis and from the blood of another patient treated with extracorporeal membrane oxygenation. Susceptibility studies were performed on these three isolates and another 29 isolates that colonized or caused infection in a total of 19 patients seen over the last decade. All isolates had low minimum inhibitory concentration (MIC) values for amphotericin B (MIC90 of < or = 0.02 microgram/ml) and flucytosine (MIC90 of 0.2 microgram/ml), and a broader range of MIC values for itraconazole (MIC90 of 0.8 microgram/ml) and fluconazole (MIC90 of 4 micrograms/ml). A colorimetric method using Alamar blue reagent showed good concordance with the standard broth macrodilution method for amphotericin B, flucytosine, and fluconazole, but less good concordance for itraconazole. Serious infections with S. cerevisiae probably should be treated with amphotericin B, with or without the addition of flucytosine.

Torulopsis glabrata: azole susceptibilities by microdilution colorimetric and macrodilution broth assays.

Fluconazole and itraconazole MICs were determined by both the standard macrodilution method of the National Committee for Clinical Laboratory Standards and a colorimetric broth microdilution method for 140 isolates of Torulopsis (Candida) glabrata obtained over a 15-year period. Using the method of the National Committee for Clinical Laboratory Standards the MICs at which 90% of isolates are inhibited (MIC50) for all isolates were 32 and 1.6 micrograms/ml for fluconazole and itraconazole, respectively. For fluconazole, the MIC90 rose from 16 to > 64 micrograms/ml when the MIC90s for isolates collected from July 1980 to June 1991 were compared with those for isolates collected from July 1991 to March 1995. For itraconazole, the MIC90s for isolates from the same time periods were 0.8 and 3.2 micrograms/ml, respectively. Although for isolates from some non-human immunodeficiency virus-infected patients the MICs rose, most of the high MICs were found for isolates from human immunodeficiency virus-infected patients who had been extensively treated with azole drugs for thrush. The colorimetric method yielded endpoints that were more definitive; concordances within 2 dilutions for the two methods were 87% for fluconazole and 86% for itraconazole.

Use of a colorimetric system for yeast susceptibility testing.

We examined the reliability and accuracy of a colorimetric assay using Alamar Blue reagent in the performance of susceptibility tests for Candida albicans. We compared the broth macrodilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) with a macrodilution method modified with the Alamar reagent and a microdilution method modified with the Alamar reagent. The MICs of fluconazole and itraconazole for 97 isolates of C. albicans and 3 control isolates were tested. For fluconazole, the Alamar-modified broth macrodilution method yielded 94% (91 of 97) concordance within 2 dilutions compared with the NCCLS method, while the microdilution method yielded 95% (92 of 97) concordance. With Alamar-modified methods for itraconazole, broth macrodilution yielded 97% (94 of 97) concordance within 2 dilutions. MICs obtained by the microdilution method, although tightly nested, were shifted to a higher value when compared with those obtained by the NCCLS method; there was only 77% (75 of 97) concordance within 2 dilutions but 97% concordance (94 of 97) within 3 dilutions. Tests by all methods with quality control strains showed excellent reproducibilities. For fluconazole, the methods modified with the Alamar reagent yielded clear endpoints and excellent correlation for the broth macrodilution and microdilution methods. For itraconazole, the methods modified with the Alamar reagent yielded clear endpoints and were reproducible, but higher MICs were obtained by the microdilution methods compared with those obtained by the NCCLS methods.

Susceptibility of ciprofloxacin-resistant staphylococci and enterococci to clinafloxacin.

Clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis obtained from the Ann Arbor Veterans Affairs Medical Center within the last decade were tested for susceptibility to ciprofloxacin and clinafloxacin. For MRSA isolates, the minimum inhibitory concentrations (MICs) of ciprofloxacin were several fold higher than those noted with clinafloxacin. Prior to the introduction of the fluoroquinolones (1984-1985), all MRSA isolates were susceptible to ciprofloxacin and clinafloxacin. By 1993, virtually all MRSA isolates were resistant to ciprofloxacin and a 50-fold increase in the MIC50 and MIC90 for clinafloxacin was seen. In 1985-1986, most enterococcal isolates were susceptible to ciprofloxacin and clinafloxacin. By 1993, one-third of all enterococci were resistant to both ciprofloxacin and clinafloxacin. Fluoroquinolone resistance developed more quickly in enterococci that demonstrated high-level gentamicin resistance. Thus, cross-resistance between clinafloxacin and ciprofloxacin was seen; however, the lower MICs of clinafloxacin for MRSA may allow the use of this drug for some MRSA infections.

Colonization and transmission of high-level gentamicin-resistant enterococci in a long-term care facility.

OBJECTIVES: To assess the prevalence of high-level gentamicin-resistant enterococcus (HGRE) colonization, transmission patterns, and spectrum of illness among residents of a long-term care facility. DESIGN: Monthly surveillance for HGRE colonization of wounds, rectum, and perineum over a 1-year period. SETTING: A Veterans Affairs long-term care facility attached to an acute-care facility. PATIENTS: All 341 patients in the facility during the observation period. RESULTS: Over the 1-year period, 120 patients (35.2%) were colonized with HGRE at least once, with an overall monthly colonization rate of 20 +/- 1.5%. HGRE were isolated from rectum (12.8%), wounds (11.7%), and perineum (9.3%). Patients with the poorest functional status had the highest rate of colonization (P < 0.0005). HGRE-colonized patients were more likely to be colonized with methicillin-resistant Staphylococcus aureus (51% versus 25%; P < 0.0005). Seventy-four patients (21.7%) were colonized at admission or at the start of the study. Another 46 patients (13.5%) acquired HGRE during the study, including 36 who acquired HGRE while in the long-term care facility and 10 who were positive when transferred back from the acute-care hospital. Based on plasmid profiles, only two patients appeared to have isolates similar to those of current or previous roommates. Carriage of HGRE was transient in most cases. Only 20 patients were colonized for 4 or more months, and those patients usually carried different strains intermittently. Infections were infrequent, occurring in only 4.1% of total patients. CONCLUSIONS: In our long-term care facility, HGRE were endemic, and new acquisition of HGRE occurred frequently. However, only two patients had evidence of acquisition from a roommate, suggesting that cross-infection from a roommate was not a major route of spread of HGRE.

Azole resistance in oropharyngeal Candida albicans strains isolated from patients infected with human immunodeficiency virus.

For 212 oropharyngeal isolates of Candida albicans, the fluconazole MICs for 50 and 90% of strains tested were 0.5 and 16 micrograms/ml, respectively, and those of itraconazole were 0.05 and 0.2 micrograms/ml, respectively. Of 16 isolates for which fluconazole MICs were > 64 micrograms/ml, itraconazole MICs for 14 were < or = 0.8 micrograms/ml and for 2 were > 6.4 micrograms/ml. Most fluconazole-resistant strains remained susceptible to itraconazole; whether itraconazole will prove effective for refractory thrush remains to be shown.

Epidemiology of oral candidiasis in HIV-infected patients: colonization, infection, treatment, and emergence of fluconazole resistance.

PURPOSE: To study the epidemiology of oral candidiasis and the effect of treatment of thrush in human immunodeficiency virus (HIV)-infected patients. PATIENTS AND METHODS: We conducted a prospective observational study of 92 patients over 1 year, including a nonblinded, randomized treatment trial of thrush with clotrimazole troches or oral fluconazole. Oral sites were cultured monthly and when thrush occurred. Candida albicans strains were typed by contour-clamped homogeneous electric field (CHEF) electrophoresis. Changes in strains were evaluated over time and in regard to their associations with particular sites, episodes of thrush, relapse after treatment, and colonization of sexual partners. Susceptibility to fluconazole was tested and CHEF analysis was done on these strains to determine the epidemiology of fluconazole resistance. RESULTS: Yeasts colonized 84% of patients. C albicans accounted for 81% of all isolates and was separated into 34 distinct strains. Most patients had persistent carriage of 1 or 2 dominant strains of C albicans. Three couples shared strains. Nineteen different C albicans strains caused 82 episodes of thrush in 45 patients. CD4 < 200/microL was associated with development of thrush. Clinical cure rates were similar with fluconazole (96%) and clotrimazole (91%), but mycologic cure was better with fluconazole (49%) than clotrimazole (27%). Following mycologic cure, colonization recurred with the same strain 74% of the time. Colonization with Torulopsis glabrata and Saccharomyces cerevisiae increased after treatment with either drug, but these organisms were never a sole cause of thrush. In a subset of 35 patients followed for over 3 months in whom fluconazole susceptibilities were performed, minimum inhibitory concentrations (MICs) to fluconazole increased only in those on fluconazole prophylaxis. Clinical failure of fluconazole was associated with an MIC > or = 64 micrograms/mL in 3 patients, and with an MIC of 8 micrograms/mL in 1 patient. In 2 of these 4 patients, the prior colonizing strain developed fluconazole resistance. In the other 2, new resistant strains were acquired. CONCLUSIONS: Many different strains of C albicans colonize and cause thrush in patients infected with HIV. Patients are usually persistently colonized with a single strain, and recurrences following treatment are usually due to the same strain. Transmission of strains may occur between couples. Fluconazole and clotrimazole are equally effective in treating thrush, but mycologic cure occurs more often with fluconazole. Fluconazole resistance in C albicans occurs most often in patients who have low CD4 counts and are taking fluconazole prophylactically for recurrent thrush. Fluconazole resistance may occur through acquisition of a new resistant strain or by development of resistance in a previously susceptible strain.

Detection and characterization of mupirocin resistance in Staphylococcus aureus.

Fourteen mupirocin-resistant Staphylococcus aureus strains were isolated over 18 months; 12 exhibited low-level resistance, while two showed high-level resistance. Highly mupirocin-resistant strains contained a large plasmid which transferred mupirocin resistance to other S. aureus strains and to Staphylococcus epidermidis. This plasmid and pAM899-1, a self-transferable gentamicin resistance plasmid, have molecular and biologic similarities.

Attempts to eradicate methicillin-resistant Staphylococcus aureus from a long-term-care facility with the use of mupirocin ointment.

PURPOSE: To assess the impact of the use of mupirocin ointment on colonization, transmission, and infection with methicillin-resistant Staphylococcus aureus (MRSA) in a long-term-care facility. PATIENTS AND METHODS: All 321 residents of a Veterans Affairs long-term-care facility from June 1990 through June 1991 were studied for MRSA colonization and infection. MRSA-colonized patients received mupirocin ointment to nares in the first 7 months and to nares and wounds in the second 5 months. The effect of mupirocin use on MRSA colonization and infection was monitored. All S. aureus strains isolated were tested for the development of resistance to mupirocin. RESULTS: A total of 65 patients colonized with MRSA received mupirocin ointment. Mupirocin rapidly eliminated MRSA at the sites treated in most patients by the end of 1 week. Weekly maintenance mupirocin was not adequate to prevent recurrences--40% of patients had recurrence of MRSA. Overall, MRSA colonization in the facility, which was 22.7% +/- 1% prior to the use of mupirocin, did not change when mupirocin was used in nares only (22.2% +/- 2.1%), but did decrease to 11.5% +/- 1.8% when mupirocin was used in nares and wounds. Although colonization decreased, roommate-to-roommate transmission and MRSA infection rates, low to begin with, did not change when mupirocin was used. Mupirocin-resistant MRSA strains were isolated in 10.8% of patients. CONCLUSIONS: Mupirocin ointment is effective at decreasing colonization with MRSA. However, constant surveillance was required to identify patients colonized at admission or experiencing recurrence of MRSA during maintenance treatment. Long-term use of mupirocin selected for mupirocin-resistant MRSA strains. Mupirocin should be saved for use in outbreak situations, and not used over the long term in facilities with endemic MRSA colonization.

Methicillin-resistant Staphylococcus aureus: colonization and infection in a long-term care facility.

OBJECTIVE: To assess methicillin-resistant Staphylococcus aureus (MRSA) colonization, transmission, and infection over a 1-year period in a long-term care facility with endemic MRSA. DESIGN: Monthly surveillance for MRSA colonization of nares, perineum, rectum, and wounds. SETTING: Long-term care facility attached to an acute care Veterans Affairs medical center. PATIENTS: All 341 patients in the facility had monthly surveillance cultures for 1 year. OUTCOME MEASUREMENTS: Colonization and infection with MRSA. MAIN RESULTS: The monthly MRSA colonization rate was 23% +/- 1.0%; colonization occurred most commonly in the nares and wounds. Poor functional status was associated with MRSA colonization. Most patients (65%) never acquired MRSA; 25% of patients were already colonized at admission to the facility or at the start of the study, and only 10% of newly admitted patients acquired MRSA while in the facility. These latter patients acquired several different strains in a pattern of acquisition similar to that generally seen within the facility. In the course of 1 year, only nine patients who acquired MRSA had a roommate with the same phage type; no clustering was evident, and none of these patients developed infection. Nine other patients (3%) developed MRSA infection; five of these patients required hospitalization, but none died as a result of infection. CONCLUSIONS: In the long-term care facility in which our study took place, MRSA was endemic, and the infection rate was low. In such settings, the cost effectiveness of aggressive management of MRSA (widespread screening for MRSA and eradication with antimicrobial agents) needs to be assessed.