Wild-type MIC distribution and epidemiological cutoff values for Aspergillus fumigatus and three triazoles as determined by the Clinical and Laboratory Standards Institute broth microdilution methods.

Antifungal susceptibility testing of Aspergillus species has been standardized by both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Recent studies suggest the emergence of strains of Aspergillus fumigatus with acquired resistance to azoles. The mechanisms of resistance involve mutations in the cyp51A (sterol demethylase) gene, and patterns of azole cross-resistance have been linked to specific mutations. Studies using the EUCAST broth microdilution (BMD) method have defined wild-type (WT) MIC distributions, epidemiological cutoff values (ECVs), and cross-resistance among the azoles. We tested a collection of 637 clinical isolates of A. fumigatus for which itraconazole MICs were < or = 2 microg/ml against posaconazole and voriconazole using the CLSI BMD method. An ECV of < or = 1 microg/ml encompassed the WT population of A. fumigatus for itraconazole and voriconazole, whereas an ECV of < or = 0.25 microg/ml was established for posaconazole. Our results demonstrate that the WT distribution and ECVs for A. fumigatus and the mold-active triazoles were the same when determined by the CLSI or the EUCAST BMD method. A collection of 43 isolates for which itraconazole MICs fell outside of the ECV were used to assess cross-resistance. Cross-resistance between itraconazole and posaconazole was seen for 53.5% of the isolates, whereas cross-resistance between itraconazole and voriconazole was apparent in only 7% of the isolates. The establishment of the WT MIC distribution and ECVs for the azoles and A. fumigatus will be useful in resistance surveillance and is an important step toward the development of clinical breakpoints.

Correlation of MIC with outcome for Candida species tested against caspofungin, anidulafungin, and micafungin: analysis and proposal for interpretive MIC breakpoints.

The CLSI Antifungal Subcommittee followed the M23-A2 "blueprint" to develop interpretive MIC breakpoints for anidulafungin, caspofungin, and micafungin against Candida species. MICs of < or = 2 microg/ml for all three echinocandins encompass 98.8 to 100% of all clinical isolates of Candida spp. without bisecting any species group and represent a concentration that is easily maintained throughout the dosing period. Data from phase III clinical trials demonstrate that the standard dosing regimens for each of these agents may be used to treat infections due to Candida spp. for which MICs are as high as 2 microg/ml. An MIC predictive of resistance to these agents cannot be defined based on the data from clinical trials due to the paucity of isolates for which MICs exceed 2 microg/ml. The clinical data set included only three isolates from patients treated with an echinocandin (caspofungin) for which the MICs were > 2 microg/ml (two C. parapsilosis isolates at 4 microg/ml and one C. rugosa isolate at 8 microg/ml). Based on these data, the CLSI subcommittee has decided to recommend a "susceptible only" breakpoint MIC of < or = 2 microg/ml due to the lack of echinocandin resistance in the population of Candida isolates thus far. Isolates for which MICs exceed 2 microg/ml should be designated "nonsusceptible" (NS). For strains yielding results suggestive of an NS category, the organism identification and antimicrobial-susceptibility test results should be confirmed. Subsequently, the isolates should be submitted to a reference laboratory that will confirm the results by using a CLSI reference dilution method.

Interpretive breakpoints for fluconazole and Candida revisited: a blueprint for the future of antifungal susceptibility testing.

Developing interpretive breakpoints for any given organism-drug combination requires integration of the MIC distribution, pharmacokinetic and pharmacodynamic parameters, and the relationship between in vitro activity and outcome from both in vivo and clinical studies. Previously, the Subcommittee for Antifungal Testing of the Clinical and Laboratory Standards Institute (CLSI [formerly National Committee for Clinical Laboratory Standards]) proposed MIC interpretive breakpoints for fluconazole and Candida spp. These breakpoints were considered to be somewhat weak, because the clinical data supporting them came largely from mucosal infections and there were very few infections involving strains with elevated fluconazole MICs. We readdress the issue of fluconazole breakpoints for Candida by using published clinical and microbiologic data to provide further validation of the breakpoints proposed by the CLSI in 1997. We also address interpretive breakpoints for agar disk diffusion testing of fluconazole. The MIC distribution for fluconazole was determined with a collection of 13,338 clinical isolates. The overall MIC at which 90% of the isolates were inhibited was 8 microg/ml: 91% were susceptible (S) at a MIC of or= 64 microg/ml). Similar results were obtained for 2,190 isolates from randomized clinical trials. Analysis of available data for 1,295 patient-episode-isolate events (692 represented mucosal infections and 603 represented invasive infections) from 12 published clinical studies demonstrated an overall success rate of 77%, including 85% for those episodes in which the fluconazole MIC was or= 64 microg/ml) isolates. Pharmacodynamic analysis demonstrated a strong relationship between MIC, fluconazole dose, and outcome. A dose/MIC ratio of approximately 25 was supportive of the following susceptibility breakpoints for fluconazole and Candida spp.: S, MIC or= 64 microg/ml. The corresponding disk test breakpoints are as follows: S, >or=19 mm; SDD, 15 to 18 mm; R,

Correlation of MIC with outcome for Candida species tested against voriconazole: analysis and proposal for interpretive breakpoints.

Developing interpretive breakpoints for any given organism-drug combination requires integration of the MIC distribution, pharmacokinetic and pharmacodynamic parameters, and the relationship between the in vitro activity and outcome from both in vivo and clinical studies. Using data generated by standardized broth microdilution and disk diffusion test methods, the Antifungal Susceptibility Subcommittee of the Clinical and Laboratory Standards Institute has now proposed interpretive breakpoints for voriconazole and Candida species. The MIC distribution for voriconazole was determined using a collection of 8,702 clinical isolates. The overall MIC90 was 0.25 microg/ml and 99% of the isolates were inhibited at < or = 1 microg/ml of voriconazole. Similar results were obtained for 1,681 Candida isolates (16 species) from the phase III clinical trials. Analysis of the available data for 249 patients from six phase III voriconazole clinical trials demonstrated a statistically significant correlation (P = 0.021) between MIC and investigator end-of-treatment assessment of outcome. Consistent with parallel pharmacodynamic analyses, these data support the following MIC breakpoints for voriconazole and Candida species: susceptible (S), < or = 1 microg/ml; susceptible dose dependent (SDD), 2 microg/ml; and resistant (R), > or = 4 microg/ml. The corresponding disk test breakpoints are as follows: S, > or = 17 mm; SDD, 14 to 16 mm; and R, < or = 13 mm.

Determination of fungicidal activities against yeasts and molds: lessons learned from bactericidal testing and the need for standardization.

In certain unique clinical settings, the ability of the antimicrobial agent administered to kill the pathogen outright may be quite important. These situations invariably involve infection of a site not easily accessed by host defenses and/or of a structure with essential anatomic or physiologic function such as the heart (endocarditis), central nervous system (meningitis), or bone (osteomyelitis). Likewise, infections in immunosuppressed hosts, especially those who are neutropenic, are often thought to require microbicidal therapy. Proof of the cidal nature of an antimicrobial agent in vitro is tedious, complex, and fraught with error. Although several methods for assessing in vitro bactericidal activity have been standardized (NCCLS M26-A and M21-A), the clinical relevance of these determinations is questionable and the tests are performed infrequently in most laboratories. Most of the clinical data supporting the need for microbicidal therapy and testing have focused on bacterial infections. However, given the fact that most serious fungal infections occur in profoundly immunosuppressed individuals, it is generally assumed that a cidal regimen would be preferable in that setting as well. In view of this clinical concern and the perceived need to assess the fungicidal activity of a variety of agents, we considered that it would be useful to review what is known about the issues and problems in assessing bactericidal activity and the clinical utility of such measurements. Following this review, we discuss the issue of how one defines fungicidal activity in vitro and in vivo and how feasible it might be to determine the fungicidal activity of organism-drug combinations for purposes of both drug development and clinical care. Proposed methods for fungal time-kill determinations and minimal fungicidal concentration determinations are also discussed.

Antifungal susceptibility testing: practical aspects and current challenges.

Development of standardized antifungal susceptibility testing methods has been the focus of intensive research for the last 15 years. Reference methods for yeasts (NCCLS M27-A) and molds (M38-P) are now available. The development of these methods provides researchers not only with standardized methods for testing but also with an understanding of the variables that affect interlaboratory reproducibility. With this knowledge, we have now moved into the phase of (i) demonstrating the clinical value (or lack thereof) of standardized methods, (ii) developing modifications to these reference methods that address specific problems, and (iii) developing reliable commercial test kits. Clinically relevant testing is now available for selected fungi and drugs: Candida spp. against fluconazole, itraconazole, flucytosine, and (perhaps) amphotericin B; Cryptococcus neoformans against (perhaps) fluconazole and amphotericin B; and Aspergillus spp. against (perhaps) itraconazole. Expanding the range of useful testing procedures is the current focus of research in this area.

Malignant glomus tumor: a case report and review of the literature.

Glomangiosarcoma, or malignant glomus tumor, is a very rare neoplasm that when seen typically arises from a benign glomus tumor. Despite having histologic features of malignancy, these tumors usually do not metastasize. However, when metastasis occurs this disease is often fatal. We report a case of a malignant glomus tumor arising de novo on the nose of an 89-year-old white woman, and we review the literature concerning glomangiosarcomas.

In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: review of the literature.

Voriconazole (VfendTM) is a new triazole that currently is undergoing phase III clinical trials. This review summarizes the published data obtained by NCCLS methods on the in vitro antifungal activity of voriconazole in comparison to itraconazole, amphotericin B, fluconazole, ketoconazole and flucytosine. Voriconazole had fungistatic activity against most yeasts and yeastlike species (minimum inhibitory concentrations [MICs] < 2 microg/ml) that was similar or superior to those of fluconazole, amphotericin B, and itraconazole. Against Candida glabrata and C. krusei, voriconazole MIC ranges were 0.03 to 8 and 0.01 to > 4 microg/ml, respectively. For four of the six Aspergillus spp. evaluated, voriconazole MICs (< 0.03 to 2 microg/ml) were lower than amphotericin B (0.25 to 4 microg/ml) and similar to itraconazole MICs. Voriconazole fungistatic activity against Fusarium spp. has been variable. Against E oxysporum and F. solani, most studies showed MICs ranging from 0.25 to 8 microg/ml. Voriconazole had excellent fungistatic activity against five of the six species of dimorphic fungi evaluated (MIC90s < 1.0 microg/ml). The exception was Sporothrix schenckii (MIC90s and geometric mean MICs > or = 8 microg/ml). Only amphotericin B had good fungistatic activity against the Zygomycetes species (voriconazole MICs ranged from 2 to > 32 microg/ml). Voriconazole showed excellent in vitro activity (MICs < 0.03 to 1.0 microg/ml) against most of the 50 species of dematiaceous fungi tested, but the activity of all the agents was poor against most isolates of Scedosporium prolificans and Phaeoacremonium parasiticum (Phialophora parasitica). Voriconazole had fungicidal activity against most Aspergillus spp., B. dermatitidis, and some dematiaceous fungi. In vitro/in vivo correlations should aid in the interpretation of these results.

Comparison of high-performance liquid chromatographic and microbiological methods for determination of voriconazole levels in plasma.

A new selective high-performance liquid chromatography (HPLC) method with UV detection for the determination of the investigational triazole voriconazole in human plasma by using acetonitrile precipitation followed by reverse-phase HPLC on a C(18) column was compared with a simple agar well diffusion bioassay method with Candida kefyr ATCC 46764 as the assay organism. Pooled plasma was used to prepare standard and control samples for both methods. The results of analyses with spiked serum samples (run as unknowns) were concordant by the bioassay and HPLC methods, with expected values being obtained. HPLC demonstrated an improved precision (3.47 versus 12.12%) and accuracy (0.81 versus 1.28%) compared to those of the bioassay method. The range of linearity obtained by both methods (from 0.2 to 10 microg/ml for HPLC and from 0.25 to 20 microg/ml for the bioassay) includes the range of concentrations of voriconazole (from 1.2 to 4.7 microg/ml) which are considered clinically relevant. Although either methodology could be used for the monitoring of patient therapy, the smaller variability observed with HPLC compared to that observed with the bioassay favors the use of HPLC for pharmacokinetic studies.

Current and emerging azole antifungal agents.

Major developments in research into the azole class of antifungal agents during the 1990s have provided expanded options for the treatment of many opportunistic and endemic fungal infections. Fluconazole and itraconazole have proved to be safer than both amphotericin B and ketoconazole. Despite these advances, serious fungal infections remain difficult to treat, and resistance to the available drugs is emerging. This review describes present and future uses of the currently available azole antifungal agents in the treatment of systemic and superficial fungal infections and provides a brief overview of the current status of in vitro susceptibility testing and the growing problem of clinical resistance to the azoles. Use of the currently available azoles in combination with other antifungal agents with different mechanisms of action is likely to provide enhanced efficacy. Detailed information on some of the second-generation triazoles being developed to provide extended coverage of opportunistic, endemic, and emerging fungal pathogens, as well as those in which resistance to older agents is becoming problematic, is provided.

Macrolide susceptibility and beta-lactamase production among haemophilus influenzae isolates in the United States, 1996-1997.

In 1996 and 1997, 68 hospital laboratories throughout the United States determined the beta-lactamase production and susceptibility to macrolides of 1,998 isolates of Haemophilus influenzae obtained from patients with community-acquired respiratory tract infections. The MICs at which 90% of the isolates are inhibited of azithromycin, erythromycin, and clarithromycin were 4, 8, and 16 microgram/ml, respectively. By National Committee for Clinical Laboratory Standards interpretive criteria, 99 and 78% of the isolates were susceptible to azithromycin and clarithromycin, respectively. The prevalence of beta-lactamase production was 32%.

Antifungal susceptibility testing of yeasts: a brief overview.

Antifungal susceptibility testing of yeasts has lacked reproducibility because of the absence of universally accepted guidelines. The major sources of variation in susceptibility testing have been attributed to the choice of medium and pH, inoculum preparation and size, incubation temperature and time, and end-point criteria. These parameters have been addressed by the National Committee for Clinical Laboratory Standards (NCCLS) Subcommittee on Antifungal Susceptibility Testing, and proposed guidelines have recently been published. Although major advances in antifungal susceptibility testing have been achieved, particularly through the efforts of the NCCLS subcommittee, the results of studies to date do not support a correlation between the results of in vitro susceptibility testing and in vivo response. Routine antifungal susceptibility testing of yeasts should therefore be discouraged until the methodology is standardized such that it predicts clinical outcome.

Directional coronary atherectomy complications and management.

Directional coronary atherectomy provides a predictable outcome in selected cases; however, DCA may still result in significant complications in a small number of patients. Although many of the complications are similar to those associated with PTCA, some of the complications are unique or more frequently observed with DCA. These complications are often preventable with adequate case selection and appropriate technique. Because of significant differences in the atherectomy procedure compared to PTCA, the operator should recognize and understand the technical differences to prevent potentially serious complications.

Restenosis after directional coronary atherectomy.

OBJECTIVES: This study evaluates the incidence of restenosis after successful directional coronary atherectomy and identifies risk factors for restenosis. BACKGROUND: Directional coronary atherectomy has been shown to be a safe and effective treatment of obstructive coronary artery disease; however, information regarding restenosis is limited. METHODS: Between October 1986 and December 1989, 289 patients with 332 lesions were successfully treated with directional coronary atherectomy and followed up prospectively. Clinical follow-up information was available for 98% and angiographic follow-up information was obtained for 82% at approximately 6 months, or earlier if symptoms recurred. Angiograms were quantitatively analyzed. Restenosis was defined as greater than 50% stenosis at the site of intervention. RESULTS: Seventy-four percent of patients were either asymptomatic or clinically improved after the procedure. Thirty-two percent were subsequently treated by coronary artery bypass surgery (14%), percutaneous transluminal coronary angioplasty (4%) or repeat atherectomy (13%). Angiographic evidence of restenosis was observed in 42%. The restenosis rate in native coronary arteries was 31% for primary lesions and 28% and 49%, respectively, for lesions treated with one or two previous angioplasty procedures. The restenosis rate for saphenous vein grafts was 53% for primary lesions and 58% and 82%, respectively, for lesions treated with one or two previous angioplasty procedures. The median interval to angiographically documented restenosis was 133 days. A higher restenosis rate was associated with a saphenous vein graft, hypertension, a longer lesion (greater than or equal to 10 mm), a smaller vessel diameter (less than 3 mm), a noncalcified lesion and use of a smaller (6F) device. CONCLUSIONS: Restenosis remains a limitation of directional coronary atherectomy. A subset of patients with larger vessels, shorter lesions or lesions treated with a larger (7F) device may have a more favorable outcome.

Hymenolepis diminuta: one of three enteric pathogens isolated from a child.

This article describes a case of enteritis in a 3.5-yr-old child with persistent diarrhea and the isolation of three gastrointestinal pathogens, including a rare human pathogen Hymenolepis diminuta (rat tapeworm). This is the first reported case in the northeastern United States in 20 yr and demonstrates that persons living in homes infested with rodents and arthropods are at risk of acquiring this infection.

Association of Blastocystis hominis with signs and symptoms of human disease.

Purged stools from 389 patients were evaluated microscopically for the presence of Blastocystis hominis. A total of five or more B. hominis cells per 40X field were observed in 43 patients (11%), and B. hominis was the only intestinal parasite present in 23 (6%) of these patients. Of the 23 patients, 19 had symptoms which included abdominal discomfort (15 patients), anorexia (10 patients), diarrhea (9 patients), and flatus (9 patients). The remaining four patients were asymptomatic. The proportion of eosinophils in the peripheral blood ranged from 4 to 12% in 11 (58%) of the symptomatic patients. Absolute eosinophil counts were greater than 250/microliter in 8 patients and greater than 400/microliter in 5 patients. Eosinophilia was not observed in the remaining symptomatic or asymptomatic patients. This study supports the emerging concept of the role of B. hominis as an intestinal parasite causative of human disease.

Angioplasty in total coronary artery occlusion: experience in 76 consecutive patients.

The influence of multiple clinical, angiographic and technical variables on the outcome of percutaneous transluminal coronary angioplasty was evaluated in a group of 76 consecutive patients with total coronary artery occlusion. Angioplasty was performed successfully in 53% of these patients. The likelihood of successful angioplasty was favorably influenced by: 1) a history of prior myocardial infarction in the distribution of the occluded arterial segment (p = 0.03); 2) an estimated maximal duration of arterial occlusion of less than 20 weeks (p less than 0.001); and 3) a length of nonvisualized arterial segment distal to the point of occlusion of less than 1.5 cm (p = 0.03). The outcome of coronary angioplasty was not significantly influenced by the vessel involved, the location of the occlusion within an involved vessel, the morphology of the occlusion (tapered versus abrupt) or the age and sex of the patient. There were no deaths and no vascular perforations. Four patients had recurrent coronary occlusion within 24 hours of the procedure; in three of these, recurrent occlusion was successfully treated with reangioplasty and in one, emergent surgical revascularization was performed. Embolic occlusion of an arterial branch distal to the point of total coronary occlusion occurred in 4 of the 40 successfully recanalized arteries. Seventy-five percent of patients having successful recanalization of an occluded coronary artery were free of the anginal symptoms that had prompted performance of the procedure at a mean follow-up period of 7.3 months. Thus, angioplasty of a total coronary artery occlusion can be performed safely and effectively, particularly in patients with a history of prior myocardial infarction, a brief estimated duration of coronary occlusion and a short nonvisualized occluded arterial segment.

Yersinia enterocolitica: guidelines for serologic diagnosis of human infections.

In the United States the diagnosis of human infection due to Yersinia enterocolitica is usually achieved by recovery of the microorganism. Serology as a diagnostic adjunct has been used minimally because of the absence of sufficient guidelines for interpretation of agglutinin titers. The serologic response among three groups of subjects (control, febrile, and infected) was assessed, and the serologic diagnosis of Y. enterocolitica infection was found to be multifactorial. Except in well-circumscribed outbreaks, agglutinin titers must be interpreted on an individual basis, taking into consideration the patient's age and underlying disease and the antibiotics and immunosuppressive agents being administered. Other factors affecting interpretation are the prevalence of a given serogroup of Y. enterocolitica in the community, the nature of antigen used, and the awareness of prozone phenomena. Agglutinin titers in the range of 1:128 in previously normal, healthy individuals are suggestive of infection; negative or minimal titers (greater than or equal to 1:32) do not rule out yersiniosis in an infant or in an immunosuppressed patient. Serology appears to be a secondary alternative to the more definitive cultural endeavors for use in diagnosis.

Pseudomonas aeruginosa: changes in antibiotic susceptibility, enzymatic activity, and antigenicity among colonial morphotypes.

Colonial variants of Pseudomonas aeruginosa have received renewed interest because of their occurrence in sputum cultures of patients with cystic fibrosis. We encountered 11 strains of P. aeruginosa from various body sites of non-cystic fibrosis patients. The strains showed two to three colonial variants, including smooth, rough, and iridescent morphotypes that arose from subculture of a single colony of P. aeruginosa originating from a primary source. The colonial segregants differed in antibiotic susceptibility (resistance to gentamicin, carbenicillin, chloramphenicol, and tetracycline), presence or absence of exoenzymes (gelatinase and elastase), degree of proteolytic activity (caseinase), pigmentation, and antigenicity. These observations suggest that in vivo dissociation with concomitant changes in enzymatic and surface properties might greatly enhance invasiveness. Concurrent differences in antimicrobial susceptibility among the colonial variants could account in some instances for the failure of antibiotic treatment in P. aeruginosa infections in which one would anticipate a positive therapeutic response.

Recovery of Pseudomonas aeruginosa colonial dissociants on a protease detection medium.

Dialyzed brain heart infusion-skim milk agar medium facilitated the recovery of colonial dissociants of Pseudomonas aeruginosa. Differences in colonial morphology as well as in proteolytic activity were readily visualized, thus permitting facile isolation of segregating colony types for further biochemical, serological, and susceptibility studies.