Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata.

The evolution of drug resistance has a profound impact on human health. Candida glabrata is a leading human fungal pathogen that can rapidly evolve resistance to echinocandins, which target cell wall biosynthesis and are front-line therapeutics for Candida infections. Here, we provide the first global analysis of mutations accompanying the evolution of fungal drug resistance in a human host utilizing a series of C. glabrata isolates that evolved echinocandin resistance in a patient treated with the echinocandin caspofungin for recurring bloodstream candidemia. Whole genome sequencing identified a mutation in the drug target, FKS2, accompanying a major resistance increase, and 8 additional non-synonymous mutations. The FKS2-T1987C mutation was sufficient for echinocandin resistance, and associated with a fitness cost that was mitigated with further evolution, observed in vitro and in a murine model of systemic candidemia. A CDC6-A511G(K171E) mutation acquired before FKS2-T1987C(S663P), conferred a small resistance increase. Elevated dosage of CDC55, which acquired a C463T(P155S) mutation after FKS2-T1987C(S663P), ameliorated fitness. To discover strategies to abrogate echinocandin resistance, we focused on the molecular chaperone Hsp90 and downstream effector calcineurin. Genetic or pharmacological compromise of Hsp90 or calcineurin function reduced basal tolerance and resistance. Hsp90 and calcineurin were required for caspofungin-dependent FKS2 induction, providing a mechanism governing echinocandin resistance. A mitochondrial respiration-defective petite mutant in the series revealed that the petite phenotype does not confer echinocandin resistance, but renders strains refractory to synergy between echinocandins and Hsp90 or calcineurin inhibitors. The kidneys of mice infected with the petite mutant were sterile, while those infected with the HSP90-repressible strain had reduced fungal burden. We provide the first global view of mutations accompanying the evolution of fungal drug resistance in a human host, implicate the premier compensatory mutation mitigating the cost of echinocandin resistance, and suggest a new mechanism of echinocandin resistance with broad therapeutic potential.

Effects of breakpoint changes on carbapenem susceptibility rates of Enterobacteriaceae: Results from the SENTRY Antimicrobial Surveillance Program, United States, 2008 to 2012.

In the absence of clinical resistance, breakpoints for many antimicrobial agents are often set high. Clinical failures following use of the agents over time requires re-evaluation of breakpoints. This is based on patient response, pharmacokinetic/pharmacodynamic information and in vitro minimal inhibitory concentration data. Data from the SENTRY Antimicrobial Surveillance Program has shown that Clinical and Laboratory Standards Institute breakpoint changes for carbapenems that occurred between 2008 and 2012 in North America have resulted in decreased levels of susceptibility for some species. In particular, reduced susceptibility to imipenem was observed for Proteus mirabilis (35%) and Morganella morganii (80%). Minor decreases in susceptibility were also noted for Enterobacter species with ertapenem (5%) and imipenem (4.3%), and Serratia species with imipenem (6.4%). No significant decreases in susceptibility were observed for meropenem following the breakpoint changes. There were no earlier breakpoints established for doripenem. Very few of these Enterobacteriaceae produce carbapenamase enzymes; therefore, the clinical significance of these changes has not yet been clearly determined. In conclusion, ongoing surveillance studies with in vitro minimum inhibitory concentration data are essential in predicting the need for breakpoint changes and in identifying the impact of such changes on the percent susceptibility of different species.

En l'absence de résistance clinique, la résistance de nombreux antimicrobiens est souvent fixée à un seuil élevé. En raison de l'échec clinique de certains de ces médicaments, il faut en réévaluer les seuils de résistance, d'après la réponse du patient, l'information pharmacocinétique et pharmacodynamique et les données relatives à la concentration minimale inhibitrice in vitro. Les données du programme de surveillance antimicrobienne SENTRY ont révélé que les changements au seuil de résistance des carbapénèmes établis par le Clinical and Laboratory Standards Institute entre 2008 et 2012 en Amérique du Nord ont entraîné une diminution de la susceptibilité de certaines espèces. Notamment, les chercheurs ont observé une susceptibilité réduite du Proteus mirabilis (35 %) et du Morganella morganii (80 %) à l'imipénem. Ils ont également remarqué de légères diminutions de la susceptibilité des espèces d'Enterobacter à l'ertapénem (5 %) et à l'imipénem (4,3 %), ainsi que des espèces de Serratia à l'imipénem (6,4 %). La susceptibilité du méropénem n'a pas diminué de manière significative, tandis qu'aucun seuil de résistance n'avait été établi auparavant pour le doripénem. Puisque très peu de ces entérobactériacés produisent des enzymes de carbapénémase, la signification clinique de ces changements n'est pas encore claire. Bref, il est essentiel de poursuivre les études de surveillance pour colliger des données sur les concentrations minimales inhibitrices in vitro afin de prédire la nécessité de changer le seuil de résistance et de déterminer les conséquences de ces changements sur le pourcentage de susceptibilité des diverses espèces.

Current and future challenges in the development of antimicrobial agents.

Micro-organisms exist to survive. Even in the absence of antimicrobial agents, many have determinants of resistance that may be expressed phenotypically, should the need arise. With the advent of the antibiotic age, as more and more drugs were developed to treat serious infections, micro-organisms (particularly bacteria) rapidly developed resistance determinants to prevent their own demise.The most important determinants of resistance have been in the Gram-positive and Gram-negative bacteria. Among Gram-positive bacteria, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and penicillin-resistant Streptococcus pneumoniae (PRSP) have taxed researchers and pharmaceutical companies to develop new agents that are effective against these resistant strains. Among the Gram-negative bacteria, extended-spectrum beta-lactamase (ESBL) enzymes, carbapenemases (CREs) and the so-called amp-C enzymes that may be readily transferred between species of enterobacteriaceae and other facultative species have created multi-drug resistant organisms that are difficult to treat. Other resistance determinants have been seen in other clinically important bacterial species such as Neisseria gonorrhoeae, Clostridium difficile, Haemophilus influenzae and Mycobacterium tuberculosis. These issues have now spread to fungal agents of infection.A variety of modalities have been used to stem the tide of resistance. These include the development of niche compounds that target specific resistance determinants. Other approaches have been to find new targets for antimicrobial activity, use of combination agents that are effective against more than one target in the cell, or new delivery mechanism to maximize the concentration of antimicrobial agents at the site of infection without causing toxicity to the host. It is important that such new modalities have been proved effective for clinical therapy. Animal models and non-mammalian systems have been developed to determine if new agents will reach sufficient concentrations at infection sites to predict clinical efficacy without toxicity. It will also be key to consider antimicrobial stewardship as an important component of the continuing battle to prevent the development of antimicrobial resistance.

Multicenter evaluation of the new Vitek 2 Neisseria-Haemophilus identification card.

The new Neisseria-Haemophilus identification (NH) card for Vitek 2 was compared with 16S rRNA gene sequencing (16S) as the reference method for accurate identification of Neisseria spp., Haemophilus spp., and other fastidious gram-negative bacteria. Testing was performed on the Vitek 2 XL system with modified software at three clinical trial laboratories. Reproducibility was determined with nine ATCC quality control strains tested 20 times over a minimum of 10 days at all three sites. A challenge set of 30 strains with known identifications and 371 recent fresh and frozen clinical isolates were also tested. Expected positive and negative biochemical reactions were also evaluated for substrate reproducibility. All microorganisms were tested on the NH card, and all clinical and stock isolates were saved for 16S testing. All reproducibility tests yielded expected results within a 95% confidence interval. For challenge microorganisms, there was 98% overall correct identification, including 8% low discrimination, 2% incorrect identification, and 0% unidentified. For clinical strains, there was 96.5% overall correct identification, including 10.2% low discrimination, 2.7% incorrect identification, and 0.8% unidentified. The 2.7% (10/371) of clinical isolates that gave an incorrect identification consisted of 7 isolates correct to genus and 3 strains incorrect to genus. There were an additional 27 strains (primarily Neisseria species) for which the 16S identification result was different from the NH card result. These were all unclaimed species by the system. The new NH card met all performance criteria within a 95% confidence interval compared to identification of clinical isolates by 16S.

Multicenter evaluation of the Vitek 2 anaerobe and Corynebacterium identification card.

The new anaerobe and Corynebacterium (ANC) identification card for Vitek 2 was compared with a 16S rRNA gene sequencing (16S) reference method for accuracy in the identification of corynebacteria and anaerobic species. Testing was performed on a Vitek 2 XL system with modified software at three clinical trial laboratories. Reproducibility was determined with nine ATCC quality control strains that were tested 20 times over a minimum of 10 days at all three sites. A challenge set of 50 well-characterized strains and 365 recent fresh and frozen clinical isolates were included in the study. The expected positive and negative biochemical well reactions were also evaluated for substrate reproducibility. All strains were tested with the ANC card, and clinical isolates were saved for 16S rRNA gene sequencing. All reproducibility tests yielded expected results within a 95% confidence interval, except for that with Corynebacterium striatum ATCC 6940, for which identification failed at one trial site. For the challenge isolates, there was 98% correct identification, 5% low discrimination, and 2% incorrect identification, and 0% were unidentified. For clinical strains, there was 95.1% correct identification, 4.9% low discrimination, and 4.6% incorrect identification, and 0.3% were unidentified. The 4.6% (17/365) of clinical isolates that were incorrectly identified consisted of 14 isolates that were correct at the genus level and three that were incorrect at the genus level. The new ANC card met all performance criteria within a 95% confidence interval compared to the identification performance by 16S rRNA gene sequencing.

Antifungal activity of medicinal plant extracts; preliminary screening studies.

ETHNOPHARMACOLOGICAL RELEVANCE: In the setting of HIV and organ transplantation, opportunistic fungal infections have become a common cause of morbidity and mortality. Thus antifungal therapy is playing a greater role in health care. Traditional plants are a valuable source of novel antifungals. AIM OF THE STUDY: To assess in vitro antifungal activity of aqueous plant extracts. The minimum inhibitory concentrations were determined for each extract in the setting of human pathogenic fungal isolates. MATERIALS AND METHODS: Plants were harvested and identification verified. Aqueous extracts were obtained and antifungal susceptibilities determined using serial dilutional extracts with a standardized microdilution broth methodology. Twenty-three fungal isolates were cultured and exposed to the plant extracts. Five known antifungals were used as positive controls. Results were read at 48 and 72 h. RESULTS: Of the 14 plants analyzed, Fragaria virginiana Duchesne, Epilobium angustifolium L. and Potentilla simplex Michx. demonstrated strong antifungal potential overall. Fragaria virginiana had some degree of activity against all of the fungal pathogens. Alnus viridis DC., Betula alleghaniensis Britt. and Solidago gigantea Ait. also demonstrated a significant degree of activity against many of the yeast isolates. CONCLUSION: Fragaria virginiana, Epilobium angustifolium and Potentilla simplex demonstrate promising antifungal potential.

Methicillin-resistant Staphylococcus aureus with reduced susceptibility to vancomycin in Canada.

This study defines the characteristics of decreasing vancomycin susceptibility in multiple isolates of methicillin-resistant Staphylococcus aureus (MRSA) recovered from a hospitalized patient in Canada over a period of 6 months. The MICs of the isolates increased during therapy with vancomycin. The patient fractured her right hip while in the United States. She was started on treatment with vancomycin. The MICs of successive isolates increased from < or =1 to 4 mg/L over 6 months. Then, an isolate tested at 8 mg/L initially and 4 mg/L with confirmatory E-test (AB BIODISK, Solna, Sweden). One month later, MRSA was still present in her wound, and therapy was changed to linezolid with rifampin. Subsequent cultures were negative for MRSA. Susceptibility testing was performed on the BD Phoenix (Becton Dickinson Diagnostic Systems, Sparks, MD), Dade Microscan (Dade Behring Microscan, Sacramento, CA), Pasco MIC (Becton Dickinson, Sparks, MD), Vitek 2 (bioMerieux, St. Louis, MO), and Sensititre (Trek Diagnostic Systems, Cleveland, OH) systems, and by E-test. Molecular typing (pulsed-field gel electrophoresis [PFGE]) was used to verify the relatedness of the isolates. Transmission electron microscopy (TEM) was used to assess the cell wall thickness of isolates with differing MICs. Population analysis was performed to assess for vancomycin hetero-resistance. MICs of 4 mg/L were only obtained with BD Phoenix, E-test, and broth microdilution. All isolates were identical by PFGE. The most resistant isolate had a thicker cell wall on TEM. Vancomycin hetero-resistance was observed in the resistant isolates. This is the first strain of MRSA with reduced susceptibility to vancomycin reported in Canada. The breakpoints for vancomycin susceptibility have been revised in light of such observations.

Comparison of Uriswab to alternative methods for urine culture collection and transport: confirmation of standard culture methodology for investigation of urinary tract infections.

The ability to isolate and identify causative agents of urinary tract infections relies primarily on the quality of the urine sample that is submitted to the microbiology. The most important factors are the method of collection, the maintenance of viability of the potential pathogens during transport, and standardization of the culturing of the urine sample. This report is a composite of several investigations comparing collection and transport on urine culture paddles, with a preservative urine sponge (Uriswab), and a comparison of Uriswab with the BD preservative transport tube as methods of preservation of urinary pathogens. Primary studies showed that Uriswab maintained significantly more urinary pathogens than the urine culture paddle with fewer mixed or contaminated cultures. The two preservative transport systems were comparable for maintenance of viability of the pathogens, but there were fewer mixed cultures when samples were collected with Uriswab. This study confirms the importance of a standard volume of 1 µL of urine for culture.

Antimicrobial resistance in Haemophilus influenzae: how can we prevent the inevitable? Commentary on antimicrobial resistance in H. influenzae based on data from the TARGETed surveillance program.

Haemophilus influenzae is an important cause of respiratory tract infections, particularly in elderly persons. It is the major bacterial pathogen in acute exacerbations of chronic bronchitis (AECB) and also causes otitis media and sinusitis. In many cases, treatment is empiric, and there is a lack of understanding of resistance issues with this bacterium. There is little understanding of the epidemiology of H. influenzae respiratory infections, although some strains may be replaced by new strains that cause more severe infections. There is almost no information on how these bacteria may spread in the community. Ampicillin resistance is significant (it may be >30%), and there are few oral agents capable of reducing organism burden. There is little understanding of the epidemiology of H. influenzae respiratory infections, and almost no information on how these bacteria may spread in the community. Recent evidence suggests that these bacteria may behave in a similar way to Streptococcus pneumoniae. If that proves correct, then it will be important to follow these organisms in the community to determine if resistance determinants may spread more widely than we have thus far believed. The implications for treatment, infection prevention and control, and public health should not be underestimated as it has been with other organisms such as S. pneumoniae and Staphylococcus aureus.

Clinical, microbiological, and epidemiological findings of an outbreak of Mycobacterium abscessus hand-and-foot disease.

In 2003, we identified an outbreak of clinically distinct lesions involving the hands and feet associated with a public wading pool in Edmonton, Alberta, Canada. A total of 85 cases were identified. The management and follow-up of 41 children and 1 adult patients is presented. Skin lesions occurred within a median incubation period of 29 days and approximately 88 days for the adult patient. Lesions resolved within a median of 58 days and approximately 150 days for the adult patient. Patients were treated with clarithromycin, topical antibiotic dressings, and/or incision and drainage of pustules or followed without treatment. All resolved without complication. The pool was closed and cleaned. The M. abscessus hand-and-foot disease is characterized by the onset, mainly in children, of tender, erythematous papules, pustules, and abscesses with a self-limited course. This is the first documented M. abscessus outbreak associated with wading pool exposure.

Occurrence and antimicrobial susceptibility patterns of pathogens isolated from skin and soft tissue infections: report from the SENTRY Antimicrobial Surveillance Program (United States and Canada, 2000).

A total of 1,404 bacterial isolates were recovered from skin and soft tissue infections (SSTIs) from hospitalized patients in 24 sites in the United States (US) and 5 Canadian medical centers as part of the SENTRY Antimicrobial Surveillance Program. Isolates were collected between October and December, 2000. The rank order of pathogens was: Staphylococcus aureus (45.9%), Pseudomonas aeruginosa (10.8%), Enterococcus spp. (8.2%), Escherichia coli (7.0%), Enterobacter spp. (5.8%) and Klebsiella spp. (5.1%). The same order was observed in the US and Canada. Of note, almost 30% of S. aureus were oxacillin-resistant. Vancomycin resistance among enterococci was low (7.8%) representing a marked decrease from earlier SENTRY Program reports. Several antimicrobial agents remained very active against P. aeruginosa and Enterobacteriaceae isolates. In particular amikacin, cefepime, and the carbapenems (imipenem and meropenem) showed an excellent spectrum of activity (>95% susceptible). Extended-spectrum beta-lactamase production was observed in both E. coli (7.1%) and Klebsiella spp. (11.3%). Cefepime remained highly active, even against ceftazidime-resistant isolates of Enterobacter spp. The results of this study have identified the most common causes of SSTIs in hospitalized patients in North America, and can be used to make informed decisions concerning standards of empiric treatment for SSTIs in this region.

Susceptibility of human isolates of Salmonella typhimurium DT 104 to antimicrobial agents used in human and veterinary medicine.

Multiple antibiotic resistance is frequently observed among strains of Salmonella typhimurium DT104. We examined the antibiotic resistance patterns of 240 human isolates submitted from central and northern Alberta to our laboratory for confirmatory testing during 1996-1999. Broth microdilution MIC panels included antibiotics proposed by the Canadian National Enteric Disease Surveillance Committee for human and animal isolates. Seven different susceptibility patterns were observed. The two most common patterns accounted for 83% of isolates; 48% were susceptible to all antibiotics tested and 35% were resistant to ampicillin, tetracycline, chloramphenicol and amoxicillin-clavulanate. All strains were susceptible to enrofloxacin and trovafloxacin with variable resistance to kanamycin and chloramphenicol. There were more susceptible isolates observed in 1996 and 1997 than in 1998 and 1999, but multiple resistant isolates were found throughout the study period.

Evaluation of a no-dressing intervention for tunneled central venous catheter exit sites.

This study tested whether central venous catheter (CVC)-related sepsis could be reduced by removing a hypothesized reservoir for pathogens, the CVC exit site dressing. Seventy-eight individuals with cancer, stratified for gender (37 men and 41 women) and transplant status, with newly inserted CVCs were recruited and randomly assigned to receive either a gauze dressing or no dressing, once their catheter insertion site had healed (3 weeks). Because there was no difference in CVC-related septic episodes based on gender or transplant status, the stratification was not maintained for remaining analyses. Although there was no significant difference in CVC-related septic episodes (P =.28) or rehospitalization rates (P =.41) because of CVC-related sepsis between the dressing and no-dressing group, individuals in the dressing group developed CVC-related sepsis sooner (P =.02) than did individuals in the no-dressing group.

Outcome of bacteremia and fungemia in paediatric oncology patients.

OBJECTIVE: To determine the outcome of paediatric oncology patients with positive blood cultures. DESIGN: Retrospective chart review. SETTING: Tertiary care hospital. POPULATION STUDIED: Oncology patients up to 17 years of age with positive blood cultures from January 1, 1994 to March 31, 1999. MAIN RESULTS: There were 121 episodes of positive blood cultures in 76 patients. Seventeen episodes were excluded because blood cultures were contaminated. Of the organisms grown from the remaining episodes, 63% were Gram-positive organisms, 23% were Gram-negative organisms, 3% were fungal and 11% were mixed. There were 13 episodes with pure or mixed isolates of Staphylococcus aureus, of which nine occurred within 14 days of the placement of a new central venous tunnelled catheter. Central venous tunnelled catheters were retained in 76 of the 102 episodes when they were present. There were two relapses, and four children were admitted to the intensive care unit with septic shock, but all survived. CONCLUSIONS: The outcome was excellent with the current management of possible bacteremia in paediatric oncology patients, but the high incidence of S aureus bacteremia suggests that empirical antibiotics should be altered if sepsis is suspected within 14 days of the placement of a central venous catheter.

Factors influencing broth microdilution antimicrobial susceptibility test results for dalbavancin, a new glycopeptide agent.

Performance of antimicrobial susceptibility tests with new agents requires careful consideration of the properties of the antimicrobial to ensure that the tests are standardized, reproducible, and reflect the true potency of the drug. Dalbavancin is a new glycopeptide with potent activity against gram-positive bacterial species. The investigations described here demonstrated that methodologic modifications of procedures are necessary to ensure consistent test results, both for quality control and for routine testing of clinical isolates. Dimethyl sulfoxide is the preferred primary solvent. The addition of 0.002% polysorbate-80 (a surfactant) to dalbavancin-containing wells in the reference broth microdilution assay resulted in consistent and reproducible MIC results for three quality control strains: Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and Streptococcus pneumoniae ATCC 49619. The same degree of consistency was observed among clinical isolates of gram-positive bacterial species tested in several clinical laboratories. These results indicate that the addition of 0.002% (final concentration) of the surfactant in broth microdilution tests produces optimal dalbavancin MICs required for accurate and reproducible clinical laboratory tests, without untoward influences of substrate binding or media constituents.

Susceptibility of Staphylococcus aureus to fusidic acid: Canadian data.

BACKGROUND: Ongoing antimicrobial surveillance is important to ensure proper management of infectious diseases. There are inherent issues in estimating the relevant incidence of antimicrobial resistance from surveillance data and special issues for topical preparations. OBJECTIVE: To perform semiannual surveillance of fusidic acid susceptibility of Staphylococcus aureus strains in a Canadian tertiary care hospital. METHODS: S. aureus strains were collected twice yearly from routine cultures. Routine antimicrobial susceptibility testing was performed by an automated method. Fusidic acid susceptibility testing was performed by disk diffusion. RESULTS: Between 1999 and 2005, 2,302 S. aureus strains were tested, of which 240 were methicillin resistant (MRSA). Among all strains tested, 65 (2.8%) were resistant to fusidic acid. Ten of the MRSA strains (4.2%) were resistant to fusidic acid. Although from different patients, these were shown to be part of a hospital outbreak and were epidemiologically linked. CONCLUSIONS: There has been no trend toward increasing fusidic acid resistance in our hospital over this period.

Comparison of dalbavancin MIC values determined by Etest (AB BIODISK) and reference dilution methods using gram-positive organisms.

While standardized microdilution testing methodologies and quality control ranges exist for the novel glycolipopeptide dalbavancin, no testing methods have been described that are immediately available for routine use in clinical laboratories. In this study, we found that the dalbavancin Etest (AB BIODISK, Solna, Sweden) procedure demonstrated a high degree of agreement (100% within +/-2 log(2) dilution steps) with the standardized broth microdilution method, validating the use of the Etest as an alternative test for investigational or clinical purposes following regulatory approval.

Comparative evaluation of a new fluorescent carboxyfluorescein diacetate-modified microdilution method for antifungal susceptibility testing of Candida albicans isolates.

This report presents a fluorescent carboxyfluorescein diacetate (CFDA)-modified microdilution method used for the susceptibility testing of Candida albicans to amphotericin B, fluconazole, ketoconazole, itraconazole, voriconazole, and flucytosine. Four different broth microdilution susceptibility testing methods were simultaneously evaluated at 24 and 48 h. The MICs determined using the CFDA-modified method (MIC(cfda)) were compared to those obtained by the standard broth microdilution method (MIC(visual)) and a procedure employing the indicator Alamar blue (MIC(alamar)). The reference MIC was determined visually as recommended by the NCCLS M27-A protocol, and then quantified spectrophotometrically following agitation (MIC(spec)). The CFDA-modified microdilution method was demonstrated to effectively determine the MICs for all the antifungal drugs tested at both 24 and 48 h. The results from both the MIC(spec) and MIC(cfda) methods yielded >80% agreement within +/-1 dilution and >90% agreement within +/-2 dilutions at 24 h in comparison to the reference MIC(visual) method, respectively. The trailing growth phenomenon that occurs with azole antifungal drugs and many strains of C. albicans did not inhibit the effectiveness of the MIC(spec) and MIC(cfda) methods. The MIC(spec) and MIC(cfda) methods shared 92.8% agreement within +/-1 dilution at 24 h and 87.6% agreement within +/-1 dilution at 48 h.

Sublethal injury and resuscitation of Candida albicans after amphotericin B treatment.

Amphotericin B treatment was previously shown to inhibit Candida albicans reproduction and reduce the fluorescence of vitality-specific dyes without causing a corresponding increase in the fluorescence of the mortality-specific dyes bis-(1,3-dibutylbarbituric acid)trimethine oxonol and SYBR Green I. In the present study, we have confirmed these results and have shown that the numbers of CFU are reduced by 99.9% by treatment with 0.5 micro g of amphotericin B per ml for 10 h at 35 degrees C. This reduction was not due to fungal cell death. First, the level of reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide increased in the presence of concentrations of amphotericin B that caused greater than 90% reductions in the numbers of CFU. Second, fungal cells treated with amphotericin B at a concentration of 0.5 micro g/ml were resuscitated by further incubation at 22 degrees C for 15 h in the continued presence of amphotericin B. Third, recovery of the ability to replicate was prevented by sequential treatment with 20 micro g of miconazole per ml, which also increased the fluorescence of mortality-specific dyes to near the maximal levels achieved with 0.9 micro g of amphotericin B per ml. Sequential treatment with fluconazole and flucytosine did not increase the levels of staining with the mortality-specific dyes. Itraconazole was less effective than ketoconazole, which was less effective than miconazole. The practice of equating the loss of the capacity of C. albicans to form colonies with fungal cell death may give incorrect results in assays with amphotericin B, and the results of assays with caution with other antifungal agents that are lipophilic or that possess significant postantifungal effects may need to be interpreted.