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Current concepts in antifungal susceptibility testing, part I
Vol. 25, No. 23
December 1,2003
Current Concepts In Antifungal
Susceptibility
Testing, Part I*
Robert S. Liao, Ph.D., and W Michael Dunne, Ph.D., Division of Laboratory Medicine, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO
Abstract A variety of antifungal agents are available to treat serious fungal infections, and a number of antifungal agents are under development. With the increasing prevalence of mycotic infections, particularly in immunosuppressed patients, and with the emergence of drug resistance to these agents, there is an increased need to perform antifungal susceptibility testing to determine which of the available antifungal agents are likely to be effective therapeutically. Part I of this two-part article will address some of the major issues and concerns, as well as propose guidelines for performing yeast susceptibility testing. Part II of this article will focus on the issues related to the performance of susceptibility testing for filamentous fungi. -._____ .-___ ..-.-.” .--._. __________ --____.---.__--_____l~ --.
The Antifungal
Agents
The number and variety of classes of antifungal agents currently available or under development to treat serious infections have never been greater. The demand for standardized antifungal susceptibility testing is a response to the increased availability of systemic antifungal agents and the increase in opportunistic fungal infections. Until recently, the only systemic antifungal agents available for use were amphotericin B, ketoconazole, itraconazole, fluconazole, and 5-flucytosine (5FC). New secondgeneration triazole agents with extended spectrums of activity (voriconazole, ravuconazole, and posaconazole) and the echinocandin agents (caspofungin, anidulafungin, and micafungin) have been introduced or are under investigation. The systemic antifungal agents *Editor’s Note: Part II of this article, along with the complete reference citations, will be published in the December IS, 2003 issue of CMN (vol. 25, no. 24). Mailing Address: Robert S. Liao, Ph.D., Division of Laboratory Medicine, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63310. E-mail: robliao@ pathboxwustkedu
can be categorized on the basis of their inhibitory targets: (i) amphotericin B (membrane integrity), (ii) the azoles (ergosterol synthesis), (iii) 5-FC (nucleic acid synthesis), and (iv) the echinocandins (1,3-P-D-glucan synthesis).
Overview
of Resistance
Primary (intrinsic) resistance to antifungal agents in emerging opportunistic fungal pathogens and secondary (developed) resistance in formerly susceptible clinical isolates are causes for concern (l-4). Species-specific differences in antifungal susceptibility do exist and are impo~ant to recognize (5-8). Primary drug resistance occurs in Cryptococcus neoformans with the echinocandins and 5-FC (9). For Candida spp., primary resistance to 5-FC is very uncommon, with the exception of Candida krusei (10). Primary resistance to fluconazole is well documented in C. krusei (1 Oa) and Candida glabrata (11). Most filamentous fungi, with the exception of dermatophytes other than Microsporum gypseum, are resistant to fluconazole (12). Fusarium spp., are resistant in vitro to all the azole agents, including the newer triazole generation (13). Primary resistance to amphotericin B should be suspected in Candida
lusitaniae, Candida guilliermondii, Aspergillus terreus, Trichosporon asahii, Scedosporium spp., Scopulariopsis spp., and Fusarium spp. (14). The incidence of secondary amphotericin B resistance in Candida is very rare (15” 17) but may also be underestimated due to suboptimal susceptibility testing methods ( 18). Although the majority of ~andida albicans isolates are susceptible to the azole antifungal agents, secondary resistance to fluconazole can develop in HIV patients on long-term suppressive therapy for oroph~ngeal candidiasis and in immunosuppressed patients receiving prophylaxis for systemic mycoses (19,20). Secondary resistance to fluconazole is the most common form of resistance in C. glabrata (1 I),
In This Issue Current Concepts In Antifungal Susceptibility Testing, Part I . . . . 177 Blastomyces dermatitidis
Pneumonia in an Adolescent . . . .181 A Case Report
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and prolonged use of ketoconazole or fluconazole may give rise to C. glabruta infections (5). The SENTRY Antifungal Surveillance Program has shown that the itraconazole MlCs for bloodstream isolates of Cundidn tropicalis and Caadida paraps~~o~isfrom 1997 to 2000 are higher, while the fluconazole and itraconazole MICs for isolates of C. glabmta and C. krusei are higher (5). Posaconazole, ravuconazole, and voriconazole have been shown to be highly active against all species of Car~did~ in vitro (5); however, a decrease in the activities of both ravuconazole and voriconazole has been observed in isolates of C. glabra~a and C. tropicalis resistant to fluconazole (21). Several small studies have also recently shown that secondary resistance to itraconazole can develop in Aspergillus jiimigatus, although the frequency is believed to be low (22,23).
static (ph~acokinetics), and (iii) the test inoculum size is probably at variance with the infectious load (27). The determination of the in vivo efficacy of antifungal agents thus takes into account both host defenses and intrinsic antimicrobial activity (28). As a result, a distinction is made between clinical resistance observed in vivo and microbiological resistance observed in vitro. This distinction is important because life-threatening systemic fungal infections are often diseases of the immunocompromised, in whom the underlying condition and iatrogenic factors contribute more to the final outcome than antifungal therapy. Nevertheless, despite the fact that antifungal susceptibility testing may not always identify those patients who will respond to antifungal therapy, in vitro microbiological resistance can often be used to predict therapeutic failure (29,30).
Antifungal
NCCLS M27 Method for Yeast Susceptibility Testing
Susceptibility
Testing
The rationale for and design of in vitro antifungal susceptibility testing are similar to those for testing antibacterial agents (3). Basically, in vitro susceptibility tests are meant to provide (i) a correlation between in vivo activity and therapeutic outcome, (ii) a measure of the relative activities of two or more ~tifungal agents, and (iii) a means to monitor the development of drug resistance (24,25). It must also be recognized that there are inherent limitations with all in vitro susceptibility tests because the MIC is not a physical or chemical measurement. A second consideration is that the interaction between the microorganism and antifungal agent is artificial, and the persistence or progression of an infection can occur despite the administration of appropriate antifungal therapy (26). Susceptibility testing does not reproduce in vivo conditions, in part because (i) host factors are absent, (ii) the agent concentration is essentially
The NCCLS Subcommittee on Antifungal Susceptibility Testing published the M27-A2 reference method for broth dilution susceptibility testing of Candida spp. and C. ~eo~o~ans to allow the development of standard breakpoints to guide therapy and to decrease interlaborato~ v~iability (31). The NCCLS M27-A2 reference method is the result of a series of collaborative studies that focused on standardizing variables that influence in vitro susceptibility testing and MIC endpoint determination for yeasts, including incubation duration and temperature, medium composition, and inoculum size. MICs for yeast are thus determined after 48 h of incubation at 35°C by using RPM1 1640 medium and an inoculum size of approximately lo4 CFUlml. The M27 method involves the use of broth macrodilution or microdilution testing; the latter is less
expensive and faster to perform. This protocol has progressed from a document that examined the role of variables in standardization through proposal, tentative approval, and approval. Adherence to the M27 method has been shown to provide greater than 90% intralaboratory and interlaboratory reproducibility (32,33). These guidelines for standardization have enabled the successful undertaking of large-scale surveillance studies of the development of resistance (6mmd proficiency testing of laboratories through the College of American Pathologists (34). Standardized testing methodology should ultimately result in the development of breakpoints, with an improved understanding of the relationship between in vitro testing and in vivo outcomes. Cundida spp. and breakpoints Despite the advances brought about by adherence to the M27 methodology, limitations and problems remain. At present, the M27 methodology supports the use of testing for only a limited number of yeast species and antifungal agents. Antifungal susceptibility testing has focused largely on ~andida spp. because they are responsible for most of the fungal bloodstream infections worldwide. As a result, interpretive breakpoints continue to exist only for Candida spp. and three antifungal agents: fluconazole, itraconazole, and 5-FC. An inte~retive breakpoint is defined as the MIC that predicts clinical response to antifungal treatment through the establishment of an in vitro-in vivo correlation, The available data that correlate clinical outcome with results of Candida 5-FC susceptibility testing are limited, and breakpoints have been developed largely on the basis of animal models and historical data in combination with known clinical and
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pharmacokinetic data (4,3 1). Interpretive guidelines classify 5-FC susceptibility results into susceptible, resistant, or intermediate categories. On the other hand, antifungal susceptibility testing for azole resistance in Candida spp. is relatively well established. MIC interpretive breakpoints exist for fluconazole and itraconazole, and isolates are classified as susceptible, resistant, or susceptible-dose dependent. The susceptible-dose dependenssignation defines a breakpoi&-tliat emphasizes the importance of achieving a maximal possible concentration of fluconazole or itraconazole in blood and tissue to achieve a favorable clinical response (31). These breakpoints were established primarily on experience with oropharyngeal infections with C. albicans (3 1). As a result, the azole breakpoints may be of limited use for yeasts other than C. albicans and for the more serious, invasive infections (29-3 1).
Endpoint determination
for yeast
Endpoint determination when testing azole agents continues to be problematic, subjective, and a major source of interlaboratory variability (35). The M27-A2 method establishes the endpoint for testing of Candida spp. susceptibility to azoles at 48 h with a criterion of 80% reduction in growth (3 1). For some strains of Candida spp., persistent growth occurs over most or all of the azole concentration ranges (36). This partial inhibition of growth, also referred to as the trailing growth phenomenon or trailing endpoint, is commonly observed with the azole agents and is the single greatest cause of endpoint determination difficulties (37-39). In addition, the trailing endpoint can be exacerbated over time with some Candida isolates, producing discordant MICs of <8 pg/ml at 24 hand 264 ug/ml at 48 h (40-43). These isolates have been referred to as having a lowhigh MIC phenotype (42), because when using the NCCLS M27-A2 guidelines, such discordant MICs place these isolates into susceptible and resistant MIC interpretive categories at 24 and 48 h, respectively (3 1).
Alternatives to the NCCLS M27 Method for Yeast Susceptibility Testing An important use of the M27 reference method is to provide a standard Clinical
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basis from which other methods can be developed (31). Modifications and alternative approaches, intended to make the NCCLS methodology more objective, efficient, and convenient, have been proposed. In Europe, the European Committee on Antimicrobial Susceptibility Testing has modified the NCCLS M27 microdilution method by using a spectrophotometric endpoint determination after 24 h of incubation, supplementing the RPM1 1640 medium with 2% dextrose, and increasing the inoculum size (44). Alternative techniques and criteria have particularly focused on improving MIC endpoint determination. It is not surprising that a great many of these efforts have addressed the difficulties that exist with determining azole endpoints. It has been suggested that trailing MIC endpoints can be corrected for by changing the standardized growth conditions with an adjustment of the medium pH or composition (4 1,45). Another potential solution to the azole trailing endpoint is to decrease the incubation time to 24 h and lower the MIC endpoint to the lowest concentration of azole producing a 50% reduction in growth (29,43,46). Decreasing the incubation time for susceptibility testing to 24 h would clearly be advantageous for the clinical laboratory. However, the requirement for 48-h incubation for optimal clinical correlations may present a barrier to this change (32,43,47) and require reassessment of both MIC interpretive breakpoints and quality control ranges.
Calorimetric
adaptation
More innovative efforts have proposed using a calorimetric endpoint in a broth microdilution format by including an oxidation-reduction indicator with the yeast innoculum and drug prior to incubation (46). Different oxidationreduction indicators, such as XTT, have been used to interpret antifungal susceptibility endpoints based on the visual detection of color change (48). However, the calorimetric oxidation-reduction indicator Alamar Blue has been used most extensively and is commercially available in a broth microdilution tray called Sensititre YeastOne (TREK Diagnostics Systems Inc., Westlake, OH). The microdilution tray contains the indicator Alamar Blue and dried serial dilutions of amphotericin B, 5-FC, fluconazole, itraconazole, and 0 2003 Elsevier
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ketoconazole. Several studies have found good correlation between the NCCLS microbroth dilution method and the YeastOne calorimetric microdilution panel (35,49,50). Although improved endpoint reproducibility has been reported, the calorimetric method continues to produce trailing azole endpoints at 48 h with Candida spp. (36,46,5 1,52). The Sensititre YeastOne method appears to be a suitable alternative procedure for routine antifungal susceptibility testing of Candida spp., although species-specific discrepancies have been noted. Lower YeastOne MICs have been observed when testing C. tropicalis with fluconazole, itraconazole, and ketoconazole in comparison studies with the microdilution and macrodilution methods (52,53). Two additional calorimetric methods, called the ASTY Calorimetric Microdilution Panel and the Rapid Susceptibility Assay (RSA), have also been introduced but have not achieved broad acceptance (54,55). The RSA is unique in that it is based on suppression of glucose uptake by susceptible fungal cells in the presence of antifungal agents. However, there is poor agreement between the RSA and the M27 method for testing fluconazole and itraconazole (54).
Spectrophotometric novel adaptations
and other
A spectrophotometric adaptation of the M27 method for determining MIC endpoints has been demonstrated to provide reproducible and objective MIC endpoints (47,56,57). This simple adaptation of the M27 reference involves agitation of the microdilution plate or resuspension of the well contents using a multichannel pipettor. For the azoles, 5-FC, or caspofungin, the MIC endpoint is best defined as the lowest concentration of antifungal agent at which the absorbance is reduced to 50% in comparison to that for the drug-free growth control (47,56-58). The spectrophotometric endpoint for yeast tested with amphotericin B is an optically clear well. The spectrophotometric method correlates well with another modification of the M27 broth microdilution format that incorporates the addition of the fluorescent dye carboxyfluorescein diacetate (CFDA), used to assessfungal viability (36). Both the CFDA and spectrophotometric methods and an additional method that quantifies ergosterol 0 196.4399/00
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content in the fungal cell wall have demonstrated that the low-high phenotype strains of Candidu are in fact susceptible to the azole antifungal agents (36,40). In support of these in vitro findings, the low-high phenotype strains in a murine model of invasive candidiasis and clinical isolates from HIVinfected patients with oropharyngeal candidiasis were shown to respond clinically to fluconazole (4359). Different vitality-specific and mortality-specific fluorescent dyes have been used with flow cytometry for the rapid detection of antifungal susceptibility. Flow cytometric susceptibility testing has good correlation with the NCCLS M27 method, primarily for Candida spp. against amphotericin B and the azoles (60-63). Interestingly, the flow cytometric method may have improved sensitivity over the M27 method for the detection of amphotericin B resistance (62). Disk diffusion The agar diffusion methods (disk diffusion and Etest [AB Biodisk, Solna, Sweden]) commonly used for antibacterial testing have also been applied to antifungal susceptibility testing, but they require further validation and standardization. These agar-based methods are simpler, more economic, and discriminate contamination better than brothbased testing. The disk diffusion method for testing antifungal agents may give results comparable to those of the M27 method. However, disk diffusion has had limited application and has been applied most successfully to fluconazole and voriconazole susceptibility testing of Cundida spp. (6,64-66). Mueller-Hinton agar with 2% glucose and 5 mg of methylene blue/ml has failed to support the growth of some isolates of C. parapsilosis, C. tropicalis, and Saccharomyces cerevisiae (67) but provides more reproducible zone diameters than RPM1 1640 agar supplemented with 2% glucose (RPG) (64). Work with Candida has shown that fluconazole disk diffusion testing cannot differentiate resistant from susceptible dose-dependent isolates and may categorize some susceptible strains as resistant (66,68,69). However, a study utilizing a 25pg fluconazole disk with 495 Candida isolates and MuellerHinton agar with glucose and methylene blue found that 97% of the results 180
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were in agreement with the M27 reference method after 24 h of incubation (64). A two-fold concentration change in MICs was accompanied by a 3- to 4-mm change in the diameters of the zones of inhibition (64). Thus, disk diffusion may provide a screen for susceptible and resistant isolates, but resistant isolates must be confirmed by MIC testing. A simple agar screen assay using agar plates containing 8 mg of fluconazole/ml had 97% categorical agreement with the M27 microbroth dilution method for 100 clinical isolates of C. glabrata (66). Etest The Etest stable-agar gradient method has been demonstrated in numerous studies to be useful for determining susceptibility of Candida spp. and C. neoformans to a variety of antifungal agents. The Etest MIC is read after a 48-h incubation period as the drug concentration at which the border of the elliptical zone of inhibition intercepts the scale in the antifungal-impregnated plastic strip. The use of RPG medium appears to be optimal for most organisms and antifungal agents (6). Modified casitone agar, which has been used to minimize the trailing-endpoint phenomenon, has failed to support the growth of some isolates of C. parapsilosis, C. tropicalis, and S. cerevisiae (67). Etest strips for fluconazole, itraconazole, voriconazole, ketoconazole, amphotericin B, and 5-FC are commercially available for investigational purposes. The agreement between the Etest and the NCCLS M27-A2 method varies with different combinations of fungal species and antifungal agent. Use of the Etest and RPG medium for susceptibility testing of Candida spp. has very good agreement with the NCCLS microdilution method for fluconazole, voriconazole, posaconazole, and caspofungin (64,70-73). Of interest, the Etest may be more sensitive than the NCCLS M27-A2 method for the detection of isolates of Cundidu that are resistant to amphotericin B (16,17). In one study, approximately 30% fewer Cundida spp. isolates were inhibited by I1 .O ug of amphotericin B/ml using the Etest compared to the NCCLS M27-A2 method (74). Amphotericin B MICs of 20.38 pg/ml determined by the Etest for Cundidu spp. have been associated with thera0 2003 Elsevier Science Inc.
peutic failure in patients treated with amphotericin B for candidemia (75). In addition to the Etest, a simple modification of the NCCLS M27-A2 method using antibiotic medium 3 instead of RPM1 1640 broth has been shown to improve the detection of amphotericin B-resistant isolates (76). Unfortunately, antibiotic medium 3 is an undefined medium, which has prevented wide acceptance of its use. Establish&a clear correlation between amphot&&B MICs and outcome has been difficult when using the NCCLS M27-A2 broth dilution method (4). The M27-A2 methodology produces a narrow range of amphotericin B MIC results for most C. albicans isolates (4,74). This narrow range of MICs, between 0.25 and 1.O pg/ml, makes detecting differences in susceptibilities among isolates extremely difficult, and resistance to amphotericin B is infrequently detected (7,3 1). Most Candida spp. isolates are inhibited by 51 .Oc(g of amphotericin B/ml using the M27 method, and as a consequence, an MIC of greater than 1.O pg/ml may represent resistance (4,3 1). However, no breakpoints for amphotericin B have yet been defined (4). Several studies have shown that patients with candidemia and isolates for which the amphotericin B MICs were >0.8 yg/ml were significantly more likely to die of that infection than patients with infection due to Candida spp. for which the amphotericin B MICs were 10.8 pg/ml(77,78). The determination of minimum fungicidal concentration (MFC) values may also be useful for detecting isolates of Candida resistant to amphotericin B. A prospective study by Nguyen et al. (76) that correlated amphotericin B susceptibility results in vitro with the outcome for 105 patients with candidemia showed that the MFC range for amphotericin B was significantly broader than the MIC range and that the MFC was a better predictor of in vivo resistance. Fungicidal activity against yeasts has also been demonstrated for the echinocandin agents (79). Thus, methods based on the in vitro measurements of fungicidal activity may eventually be preferred for fungicidal antifungal agents, but standard testing guidelines are not yet available to reliably assess their clinical relevance. Clinical Microbiology
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The NCCLS M27-A2 method also includes standardized methodology for the testing of C. ~e~~~~~ff~~(80,81). The M27 microdilution method utilizes a 72-h incuba~on to test C. ~eofo~~~~ against fluconazole (3 1,82). Use of a shortened incubation time of 48 h, by using yeast nitrogen base (YNB) medium in place of RPM1 1640 and a higher inoculum, provides an overall agreement of 88% w@J&?7-A2. YNB medium produces???ider range of fluconazole MICs than the RPM1 1640 medium employed with the M27 reference method, but it is not widely used (80,81,83). The number of studies that have established the value of susceptibility testing to predict clinical response in patients with C. neoformans infections is still limited, and interpretive breakpoints have not yet been established. Secondary resistance to fluconazole can develop in C. neoformans, especially in HIV patients on long-term suppressive therapy, resulting in relapses of cryptococcal meningitis (84,85). In vitro susceptibility testing of fluconazole using the M27 method with YNB medium predicted the response to therapy in 25 HIV patients with cryptococcal meningitis; isolates with a fluconazole MIC of 216 yglml were more likely to result in the failure of therapy (1,86). The commerci~ Sensititre YeastOne calorimetric microdilution test has also been used for susceptibility testing of C. neoformans after 72 h of incubation. Comparisons of the Sensititre YeastOne method with the M27 reference proce-
Blastomyces ~~r~~~~is
dure have had variable results. At 72 h, ~phote~cin B MICs for C. neofo~ans are often lower using the YeastOne method than using the reference M27 method (52,86). In one of the larger studies, several strains that were susceptible to fluconazole and S-FC with the YeastOne method were resistant to these agents when tested by the reference M-27 broth microdilution method (86). In a recent study, the Etest was used successfully to test C. neoformans against amphotericin B, voriconazole, and fluconazole (87). For 162 clinical isolates, agreement between the Etesl and the reference M-27 method was 94% for voriconazole and 99% for amphotericin B when RPC agar and a 72-h incubation were used (87). Fluconazole MICs determined using the Etest and M-27 method showed 97% agreement for 40 clinical isolates of C. neofor~ns using RPG agar and a 48-h incubation (73). Additionally, an agar dilution susceptibility testing method using 2% Sabouraud dextrose medium containing 16 mg of fluconazole/ml was a simple and inexpensive screening method for detecting fluconazole resistance in C. neoformans (83). In that study, there was 100% agreement between the agar dilution method and the M-27 macrodilution method when testing 84 strains for which the MICs were ~16 mg/ml and 7 strains which were 216 mg/ml(88).
fungi, but some successeshave been obtained. The NCCLS procedure has been modified for susceptibility testing of Histopl~ma capsu~utum strains by increasing the inoculum size and incubation time (96 to 120 h) in order to accommodate the lower growth rate of H. capsulatum (89). In this study, failure of fluconazole therapy in patients with AIDS was determined to be more likely if the MICs for the initial isolates of H. caps&a&m were 235 pg/ml(89). Interestingly, a study that compared the susceptibility of the yeast and mycelial forms of dimorphic fungi using a modification of the M27 guidelines showed that the fluconazole MICs were lower for the yeast forms of H. capsulatum and Blastomyces dermatitidis but the same for both growth forms with itraconazole and amphotericin B (90). A study that evaluated the susceptibilities of 35 clinical isolates of the yeast Rhodotorula using the Sensititre YeastOne calorimetric microdilution method found that almost all of the isolates were resistant to fluconazole but were susceptible to the other commonly used antifungal agents (91). Another study which evaluated the susceptibilities of 30 clinical isolates of the yeast S. cerevisiae found that the azole MICs were elevated, but the broth medium RPM1 1640 did not support the growth of five isolates (92).
Other Pathogenic
Editor’s Note: Part II of this article, along with the complete reference citations, will be published in the December 1.5,2003 issue of CMN (vol. 25, no. 24).
Yeasts
There has been very limited study of the M27 method for the susceptibility testing of other non-Candida yeasts or the yeast forms of the dimorphic
Pneumonia in an Adolescent
Dennis L. Murray, M.D., FLAP, Children’s Medical Center, Medical College of Georgia, Augusta, GA; Maria J. Patterson, M.D., Ph.D, FUe Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI; Carrie Naasz, M.D., Department of Pediatrics and Human development, Michigan State University, East Lansing, MI
Introduction Within the United States, the incidence of pneumonia is reported to be Mailing Address: Dennis L. Murruy, M.D., FAAE Children’s Medical Centel: Medical Coflege of Georgia, 1446 Harper St., BG-IlOlA, Augus?a, GA 30912. Tel,: 3X-721-4725. Fax: 706-721-6832. E-mail: [email protected] Chid
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four episodes per 100 children <5 years of age and falls to 0.7 episodes per 100 children 12 to 1.5years of age (1). An etiological agent is not identified in the majority of children with communityacquired pneumonia, although the more tests that are done, the more potential causes are found (2). The case of an adolescent with a
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significant and progressive pneumonia whose sputum eventually was found to contain BEastomycesdermatitidis is presented here. The patient’s twin sister, another younger sibling, and both parents were discovered later to be ill and/or to have abnormal radiographic findings. B. dermatidis was the suspected etiology in all four cases.
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December 1,2003
Current Concepts In Antifungal
Susceptibility
Testing, Part I*
Robert S. Liao, Ph.D., and W Michael Dunne, Ph.D., Division of Laboratory Medicine, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO
Abstract A variety of antifungal agents are available to treat serious fungal infections, and a number of antifungal agents are under development. With the increasing prevalence of mycotic infections, particularly in immunosuppressed patients, and with the emergence of drug resistance to these agents, there is an increased need to perform antifungal susceptibility testing to determine which of the available antifungal agents are likely to be effective therapeutically. Part I of this two-part article will address some of the major issues and concerns, as well as propose guidelines for performing yeast susceptibility testing. Part II of this article will focus on the issues related to the performance of susceptibility testing for filamentous fungi. -._____ .-___ ..-.-.” .--._. __________ --____.---.__--_____l~ --.
The Antifungal
Agents
The number and variety of classes of antifungal agents currently available or under development to treat serious infections have never been greater. The demand for standardized antifungal susceptibility testing is a response to the increased availability of systemic antifungal agents and the increase in opportunistic fungal infections. Until recently, the only systemic antifungal agents available for use were amphotericin B, ketoconazole, itraconazole, fluconazole, and 5-flucytosine (5FC). New secondgeneration triazole agents with extended spectrums of activity (voriconazole, ravuconazole, and posaconazole) and the echinocandin agents (caspofungin, anidulafungin, and micafungin) have been introduced or are under investigation. The systemic antifungal agents *Editor’s Note: Part II of this article, along with the complete reference citations, will be published in the December IS, 2003 issue of CMN (vol. 25, no. 24). Mailing Address: Robert S. Liao, Ph.D., Division of Laboratory Medicine, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63310. E-mail: robliao@ pathboxwustkedu
can be categorized on the basis of their inhibitory targets: (i) amphotericin B (membrane integrity), (ii) the azoles (ergosterol synthesis), (iii) 5-FC (nucleic acid synthesis), and (iv) the echinocandins (1,3-P-D-glucan synthesis).
Overview
of Resistance
Primary (intrinsic) resistance to antifungal agents in emerging opportunistic fungal pathogens and secondary (developed) resistance in formerly susceptible clinical isolates are causes for concern (l-4). Species-specific differences in antifungal susceptibility do exist and are impo~ant to recognize (5-8). Primary drug resistance occurs in Cryptococcus neoformans with the echinocandins and 5-FC (9). For Candida spp., primary resistance to 5-FC is very uncommon, with the exception of Candida krusei (10). Primary resistance to fluconazole is well documented in C. krusei (1 Oa) and Candida glabrata (11). Most filamentous fungi, with the exception of dermatophytes other than Microsporum gypseum, are resistant to fluconazole (12). Fusarium spp., are resistant in vitro to all the azole agents, including the newer triazole generation (13). Primary resistance to amphotericin B should be suspected in Candida
lusitaniae, Candida guilliermondii, Aspergillus terreus, Trichosporon asahii, Scedosporium spp., Scopulariopsis spp., and Fusarium spp. (14). The incidence of secondary amphotericin B resistance in Candida is very rare (15” 17) but may also be underestimated due to suboptimal susceptibility testing methods ( 18). Although the majority of ~andida albicans isolates are susceptible to the azole antifungal agents, secondary resistance to fluconazole can develop in HIV patients on long-term suppressive therapy for oroph~ngeal candidiasis and in immunosuppressed patients receiving prophylaxis for systemic mycoses (19,20). Secondary resistance to fluconazole is the most common form of resistance in C. glabrata (1 I),
In This Issue Current Concepts In Antifungal Susceptibility Testing, Part I . . . . 177 Blastomyces dermatitidis
Pneumonia in an Adolescent . . . .181 A Case Report
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and prolonged use of ketoconazole or fluconazole may give rise to C. glabruta infections (5). The SENTRY Antifungal Surveillance Program has shown that the itraconazole MlCs for bloodstream isolates of Cundidn tropicalis and Caadida paraps~~o~isfrom 1997 to 2000 are higher, while the fluconazole and itraconazole MICs for isolates of C. glabmta and C. krusei are higher (5). Posaconazole, ravuconazole, and voriconazole have been shown to be highly active against all species of Car~did~ in vitro (5); however, a decrease in the activities of both ravuconazole and voriconazole has been observed in isolates of C. glabra~a and C. tropicalis resistant to fluconazole (21). Several small studies have also recently shown that secondary resistance to itraconazole can develop in Aspergillus jiimigatus, although the frequency is believed to be low (22,23).
static (ph~acokinetics), and (iii) the test inoculum size is probably at variance with the infectious load (27). The determination of the in vivo efficacy of antifungal agents thus takes into account both host defenses and intrinsic antimicrobial activity (28). As a result, a distinction is made between clinical resistance observed in vivo and microbiological resistance observed in vitro. This distinction is important because life-threatening systemic fungal infections are often diseases of the immunocompromised, in whom the underlying condition and iatrogenic factors contribute more to the final outcome than antifungal therapy. Nevertheless, despite the fact that antifungal susceptibility testing may not always identify those patients who will respond to antifungal therapy, in vitro microbiological resistance can often be used to predict therapeutic failure (29,30).
Antifungal
NCCLS M27 Method for Yeast Susceptibility Testing
Susceptibility
Testing
The rationale for and design of in vitro antifungal susceptibility testing are similar to those for testing antibacterial agents (3). Basically, in vitro susceptibility tests are meant to provide (i) a correlation between in vivo activity and therapeutic outcome, (ii) a measure of the relative activities of two or more ~tifungal agents, and (iii) a means to monitor the development of drug resistance (24,25). It must also be recognized that there are inherent limitations with all in vitro susceptibility tests because the MIC is not a physical or chemical measurement. A second consideration is that the interaction between the microorganism and antifungal agent is artificial, and the persistence or progression of an infection can occur despite the administration of appropriate antifungal therapy (26). Susceptibility testing does not reproduce in vivo conditions, in part because (i) host factors are absent, (ii) the agent concentration is essentially
The NCCLS Subcommittee on Antifungal Susceptibility Testing published the M27-A2 reference method for broth dilution susceptibility testing of Candida spp. and C. ~eo~o~ans to allow the development of standard breakpoints to guide therapy and to decrease interlaborato~ v~iability (31). The NCCLS M27-A2 reference method is the result of a series of collaborative studies that focused on standardizing variables that influence in vitro susceptibility testing and MIC endpoint determination for yeasts, including incubation duration and temperature, medium composition, and inoculum size. MICs for yeast are thus determined after 48 h of incubation at 35°C by using RPM1 1640 medium and an inoculum size of approximately lo4 CFUlml. The M27 method involves the use of broth macrodilution or microdilution testing; the latter is less
expensive and faster to perform. This protocol has progressed from a document that examined the role of variables in standardization through proposal, tentative approval, and approval. Adherence to the M27 method has been shown to provide greater than 90% intralaboratory and interlaboratory reproducibility (32,33). These guidelines for standardization have enabled the successful undertaking of large-scale surveillance studies of the development of resistance (6mmd proficiency testing of laboratories through the College of American Pathologists (34). Standardized testing methodology should ultimately result in the development of breakpoints, with an improved understanding of the relationship between in vitro testing and in vivo outcomes. Cundida spp. and breakpoints Despite the advances brought about by adherence to the M27 methodology, limitations and problems remain. At present, the M27 methodology supports the use of testing for only a limited number of yeast species and antifungal agents. Antifungal susceptibility testing has focused largely on ~andida spp. because they are responsible for most of the fungal bloodstream infections worldwide. As a result, interpretive breakpoints continue to exist only for Candida spp. and three antifungal agents: fluconazole, itraconazole, and 5-FC. An inte~retive breakpoint is defined as the MIC that predicts clinical response to antifungal treatment through the establishment of an in vitro-in vivo correlation, The available data that correlate clinical outcome with results of Candida 5-FC susceptibility testing are limited, and breakpoints have been developed largely on the basis of animal models and historical data in combination with known clinical and
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Clinical Microbiology
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pharmacokinetic data (4,3 1). Interpretive guidelines classify 5-FC susceptibility results into susceptible, resistant, or intermediate categories. On the other hand, antifungal susceptibility testing for azole resistance in Candida spp. is relatively well established. MIC interpretive breakpoints exist for fluconazole and itraconazole, and isolates are classified as susceptible, resistant, or susceptible-dose dependent. The susceptible-dose dependenssignation defines a breakpoi&-tliat emphasizes the importance of achieving a maximal possible concentration of fluconazole or itraconazole in blood and tissue to achieve a favorable clinical response (31). These breakpoints were established primarily on experience with oropharyngeal infections with C. albicans (3 1). As a result, the azole breakpoints may be of limited use for yeasts other than C. albicans and for the more serious, invasive infections (29-3 1).
Endpoint determination
for yeast
Endpoint determination when testing azole agents continues to be problematic, subjective, and a major source of interlaboratory variability (35). The M27-A2 method establishes the endpoint for testing of Candida spp. susceptibility to azoles at 48 h with a criterion of 80% reduction in growth (3 1). For some strains of Candida spp., persistent growth occurs over most or all of the azole concentration ranges (36). This partial inhibition of growth, also referred to as the trailing growth phenomenon or trailing endpoint, is commonly observed with the azole agents and is the single greatest cause of endpoint determination difficulties (37-39). In addition, the trailing endpoint can be exacerbated over time with some Candida isolates, producing discordant MICs of <8 pg/ml at 24 hand 264 ug/ml at 48 h (40-43). These isolates have been referred to as having a lowhigh MIC phenotype (42), because when using the NCCLS M27-A2 guidelines, such discordant MICs place these isolates into susceptible and resistant MIC interpretive categories at 24 and 48 h, respectively (3 1).
Alternatives to the NCCLS M27 Method for Yeast Susceptibility Testing An important use of the M27 reference method is to provide a standard Clinical
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25:23,2(x)3
basis from which other methods can be developed (31). Modifications and alternative approaches, intended to make the NCCLS methodology more objective, efficient, and convenient, have been proposed. In Europe, the European Committee on Antimicrobial Susceptibility Testing has modified the NCCLS M27 microdilution method by using a spectrophotometric endpoint determination after 24 h of incubation, supplementing the RPM1 1640 medium with 2% dextrose, and increasing the inoculum size (44). Alternative techniques and criteria have particularly focused on improving MIC endpoint determination. It is not surprising that a great many of these efforts have addressed the difficulties that exist with determining azole endpoints. It has been suggested that trailing MIC endpoints can be corrected for by changing the standardized growth conditions with an adjustment of the medium pH or composition (4 1,45). Another potential solution to the azole trailing endpoint is to decrease the incubation time to 24 h and lower the MIC endpoint to the lowest concentration of azole producing a 50% reduction in growth (29,43,46). Decreasing the incubation time for susceptibility testing to 24 h would clearly be advantageous for the clinical laboratory. However, the requirement for 48-h incubation for optimal clinical correlations may present a barrier to this change (32,43,47) and require reassessment of both MIC interpretive breakpoints and quality control ranges.
Calorimetric
adaptation
More innovative efforts have proposed using a calorimetric endpoint in a broth microdilution format by including an oxidation-reduction indicator with the yeast innoculum and drug prior to incubation (46). Different oxidationreduction indicators, such as XTT, have been used to interpret antifungal susceptibility endpoints based on the visual detection of color change (48). However, the calorimetric oxidation-reduction indicator Alamar Blue has been used most extensively and is commercially available in a broth microdilution tray called Sensititre YeastOne (TREK Diagnostics Systems Inc., Westlake, OH). The microdilution tray contains the indicator Alamar Blue and dried serial dilutions of amphotericin B, 5-FC, fluconazole, itraconazole, and 0 2003 Elsevier
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ketoconazole. Several studies have found good correlation between the NCCLS microbroth dilution method and the YeastOne calorimetric microdilution panel (35,49,50). Although improved endpoint reproducibility has been reported, the calorimetric method continues to produce trailing azole endpoints at 48 h with Candida spp. (36,46,5 1,52). The Sensititre YeastOne method appears to be a suitable alternative procedure for routine antifungal susceptibility testing of Candida spp., although species-specific discrepancies have been noted. Lower YeastOne MICs have been observed when testing C. tropicalis with fluconazole, itraconazole, and ketoconazole in comparison studies with the microdilution and macrodilution methods (52,53). Two additional calorimetric methods, called the ASTY Calorimetric Microdilution Panel and the Rapid Susceptibility Assay (RSA), have also been introduced but have not achieved broad acceptance (54,55). The RSA is unique in that it is based on suppression of glucose uptake by susceptible fungal cells in the presence of antifungal agents. However, there is poor agreement between the RSA and the M27 method for testing fluconazole and itraconazole (54).
Spectrophotometric novel adaptations
and other
A spectrophotometric adaptation of the M27 method for determining MIC endpoints has been demonstrated to provide reproducible and objective MIC endpoints (47,56,57). This simple adaptation of the M27 reference involves agitation of the microdilution plate or resuspension of the well contents using a multichannel pipettor. For the azoles, 5-FC, or caspofungin, the MIC endpoint is best defined as the lowest concentration of antifungal agent at which the absorbance is reduced to 50% in comparison to that for the drug-free growth control (47,56-58). The spectrophotometric endpoint for yeast tested with amphotericin B is an optically clear well. The spectrophotometric method correlates well with another modification of the M27 broth microdilution format that incorporates the addition of the fluorescent dye carboxyfluorescein diacetate (CFDA), used to assessfungal viability (36). Both the CFDA and spectrophotometric methods and an additional method that quantifies ergosterol 0 196.4399/00
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content in the fungal cell wall have demonstrated that the low-high phenotype strains of Candidu are in fact susceptible to the azole antifungal agents (36,40). In support of these in vitro findings, the low-high phenotype strains in a murine model of invasive candidiasis and clinical isolates from HIVinfected patients with oropharyngeal candidiasis were shown to respond clinically to fluconazole (4359). Different vitality-specific and mortality-specific fluorescent dyes have been used with flow cytometry for the rapid detection of antifungal susceptibility. Flow cytometric susceptibility testing has good correlation with the NCCLS M27 method, primarily for Candida spp. against amphotericin B and the azoles (60-63). Interestingly, the flow cytometric method may have improved sensitivity over the M27 method for the detection of amphotericin B resistance (62). Disk diffusion The agar diffusion methods (disk diffusion and Etest [AB Biodisk, Solna, Sweden]) commonly used for antibacterial testing have also been applied to antifungal susceptibility testing, but they require further validation and standardization. These agar-based methods are simpler, more economic, and discriminate contamination better than brothbased testing. The disk diffusion method for testing antifungal agents may give results comparable to those of the M27 method. However, disk diffusion has had limited application and has been applied most successfully to fluconazole and voriconazole susceptibility testing of Cundida spp. (6,64-66). Mueller-Hinton agar with 2% glucose and 5 mg of methylene blue/ml has failed to support the growth of some isolates of C. parapsilosis, C. tropicalis, and Saccharomyces cerevisiae (67) but provides more reproducible zone diameters than RPM1 1640 agar supplemented with 2% glucose (RPG) (64). Work with Candida has shown that fluconazole disk diffusion testing cannot differentiate resistant from susceptible dose-dependent isolates and may categorize some susceptible strains as resistant (66,68,69). However, a study utilizing a 25pg fluconazole disk with 495 Candida isolates and MuellerHinton agar with glucose and methylene blue found that 97% of the results 180
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were in agreement with the M27 reference method after 24 h of incubation (64). A two-fold concentration change in MICs was accompanied by a 3- to 4-mm change in the diameters of the zones of inhibition (64). Thus, disk diffusion may provide a screen for susceptible and resistant isolates, but resistant isolates must be confirmed by MIC testing. A simple agar screen assay using agar plates containing 8 mg of fluconazole/ml had 97% categorical agreement with the M27 microbroth dilution method for 100 clinical isolates of C. glabrata (66). Etest The Etest stable-agar gradient method has been demonstrated in numerous studies to be useful for determining susceptibility of Candida spp. and C. neoformans to a variety of antifungal agents. The Etest MIC is read after a 48-h incubation period as the drug concentration at which the border of the elliptical zone of inhibition intercepts the scale in the antifungal-impregnated plastic strip. The use of RPG medium appears to be optimal for most organisms and antifungal agents (6). Modified casitone agar, which has been used to minimize the trailing-endpoint phenomenon, has failed to support the growth of some isolates of C. parapsilosis, C. tropicalis, and S. cerevisiae (67). Etest strips for fluconazole, itraconazole, voriconazole, ketoconazole, amphotericin B, and 5-FC are commercially available for investigational purposes. The agreement between the Etest and the NCCLS M27-A2 method varies with different combinations of fungal species and antifungal agent. Use of the Etest and RPG medium for susceptibility testing of Candida spp. has very good agreement with the NCCLS microdilution method for fluconazole, voriconazole, posaconazole, and caspofungin (64,70-73). Of interest, the Etest may be more sensitive than the NCCLS M27-A2 method for the detection of isolates of Cundidu that are resistant to amphotericin B (16,17). In one study, approximately 30% fewer Cundida spp. isolates were inhibited by I1 .O ug of amphotericin B/ml using the Etest compared to the NCCLS M27-A2 method (74). Amphotericin B MICs of 20.38 pg/ml determined by the Etest for Cundidu spp. have been associated with thera0 2003 Elsevier Science Inc.
peutic failure in patients treated with amphotericin B for candidemia (75). In addition to the Etest, a simple modification of the NCCLS M27-A2 method using antibiotic medium 3 instead of RPM1 1640 broth has been shown to improve the detection of amphotericin B-resistant isolates (76). Unfortunately, antibiotic medium 3 is an undefined medium, which has prevented wide acceptance of its use. Establish&a clear correlation between amphot&&B MICs and outcome has been difficult when using the NCCLS M27-A2 broth dilution method (4). The M27-A2 methodology produces a narrow range of amphotericin B MIC results for most C. albicans isolates (4,74). This narrow range of MICs, between 0.25 and 1.O pg/ml, makes detecting differences in susceptibilities among isolates extremely difficult, and resistance to amphotericin B is infrequently detected (7,3 1). Most Candida spp. isolates are inhibited by 51 .Oc(g of amphotericin B/ml using the M27 method, and as a consequence, an MIC of greater than 1.O pg/ml may represent resistance (4,3 1). However, no breakpoints for amphotericin B have yet been defined (4). Several studies have shown that patients with candidemia and isolates for which the amphotericin B MICs were >0.8 yg/ml were significantly more likely to die of that infection than patients with infection due to Candida spp. for which the amphotericin B MICs were 10.8 pg/ml(77,78). The determination of minimum fungicidal concentration (MFC) values may also be useful for detecting isolates of Candida resistant to amphotericin B. A prospective study by Nguyen et al. (76) that correlated amphotericin B susceptibility results in vitro with the outcome for 105 patients with candidemia showed that the MFC range for amphotericin B was significantly broader than the MIC range and that the MFC was a better predictor of in vivo resistance. Fungicidal activity against yeasts has also been demonstrated for the echinocandin agents (79). Thus, methods based on the in vitro measurements of fungicidal activity may eventually be preferred for fungicidal antifungal agents, but standard testing guidelines are not yet available to reliably assess their clinical relevance. Clinical Microbiology
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The NCCLS M27-A2 method also includes standardized methodology for the testing of C. ~e~~~~~ff~~(80,81). The M27 microdilution method utilizes a 72-h incuba~on to test C. ~eofo~~~~ against fluconazole (3 1,82). Use of a shortened incubation time of 48 h, by using yeast nitrogen base (YNB) medium in place of RPM1 1640 and a higher inoculum, provides an overall agreement of 88% w@J&?7-A2. YNB medium produces???ider range of fluconazole MICs than the RPM1 1640 medium employed with the M27 reference method, but it is not widely used (80,81,83). The number of studies that have established the value of susceptibility testing to predict clinical response in patients with C. neoformans infections is still limited, and interpretive breakpoints have not yet been established. Secondary resistance to fluconazole can develop in C. neoformans, especially in HIV patients on long-term suppressive therapy, resulting in relapses of cryptococcal meningitis (84,85). In vitro susceptibility testing of fluconazole using the M27 method with YNB medium predicted the response to therapy in 25 HIV patients with cryptococcal meningitis; isolates with a fluconazole MIC of 216 yglml were more likely to result in the failure of therapy (1,86). The commerci~ Sensititre YeastOne calorimetric microdilution test has also been used for susceptibility testing of C. neoformans after 72 h of incubation. Comparisons of the Sensititre YeastOne method with the M27 reference proce-
Blastomyces ~~r~~~~is
dure have had variable results. At 72 h, ~phote~cin B MICs for C. neofo~ans are often lower using the YeastOne method than using the reference M27 method (52,86). In one of the larger studies, several strains that were susceptible to fluconazole and S-FC with the YeastOne method were resistant to these agents when tested by the reference M-27 broth microdilution method (86). In a recent study, the Etest was used successfully to test C. neoformans against amphotericin B, voriconazole, and fluconazole (87). For 162 clinical isolates, agreement between the Etesl and the reference M-27 method was 94% for voriconazole and 99% for amphotericin B when RPC agar and a 72-h incubation were used (87). Fluconazole MICs determined using the Etest and M-27 method showed 97% agreement for 40 clinical isolates of C. neofor~ns using RPG agar and a 48-h incubation (73). Additionally, an agar dilution susceptibility testing method using 2% Sabouraud dextrose medium containing 16 mg of fluconazole/ml was a simple and inexpensive screening method for detecting fluconazole resistance in C. neoformans (83). In that study, there was 100% agreement between the agar dilution method and the M-27 macrodilution method when testing 84 strains for which the MICs were ~16 mg/ml and 7 strains which were 216 mg/ml(88).
fungi, but some successeshave been obtained. The NCCLS procedure has been modified for susceptibility testing of Histopl~ma capsu~utum strains by increasing the inoculum size and incubation time (96 to 120 h) in order to accommodate the lower growth rate of H. capsulatum (89). In this study, failure of fluconazole therapy in patients with AIDS was determined to be more likely if the MICs for the initial isolates of H. caps&a&m were 235 pg/ml(89). Interestingly, a study that compared the susceptibility of the yeast and mycelial forms of dimorphic fungi using a modification of the M27 guidelines showed that the fluconazole MICs were lower for the yeast forms of H. capsulatum and Blastomyces dermatitidis but the same for both growth forms with itraconazole and amphotericin B (90). A study that evaluated the susceptibilities of 35 clinical isolates of the yeast Rhodotorula using the Sensititre YeastOne calorimetric microdilution method found that almost all of the isolates were resistant to fluconazole but were susceptible to the other commonly used antifungal agents (91). Another study which evaluated the susceptibilities of 30 clinical isolates of the yeast S. cerevisiae found that the azole MICs were elevated, but the broth medium RPM1 1640 did not support the growth of five isolates (92).
Other Pathogenic
Editor’s Note: Part II of this article, along with the complete reference citations, will be published in the December 1.5,2003 issue of CMN (vol. 25, no. 24).
Yeasts
There has been very limited study of the M27 method for the susceptibility testing of other non-Candida yeasts or the yeast forms of the dimorphic
Pneumonia in an Adolescent
Dennis L. Murray, M.D., FLAP, Children’s Medical Center, Medical College of Georgia, Augusta, GA; Maria J. Patterson, M.D., Ph.D, FUe Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI; Carrie Naasz, M.D., Department of Pediatrics and Human development, Michigan State University, East Lansing, MI
Introduction Within the United States, the incidence of pneumonia is reported to be Mailing Address: Dennis L. Murruy, M.D., FAAE Children’s Medical Centel: Medical Coflege of Georgia, 1446 Harper St., BG-IlOlA, Augus?a, GA 30912. Tel,: 3X-721-4725. Fax: 706-721-6832. E-mail: [email protected] Chid
Microbiology
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25:23,2003
four episodes per 100 children <5 years of age and falls to 0.7 episodes per 100 children 12 to 1.5years of age (1). An etiological agent is not identified in the majority of children with communityacquired pneumonia, although the more tests that are done, the more potential causes are found (2). The case of an adolescent with a
0 2003 Else-&r
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significant and progressive pneumonia whose sputum eventually was found to contain BEastomycesdermatitidis is presented here. The patient’s twin sister, another younger sibling, and both parents were discovered later to be ill and/or to have abnormal radiographic findings. B. dermatidis was the suspected etiology in all four cases.
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