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`CLINICAL MICROBIOLOGY REVIEWS
`
`VOLUME 6 I OCTOBER 1993 I NUMBEFI4
`
`Josephine A. Morello, Editor in Ci1ief(1997')
`Ul'l.i1-’€l'Sii}' of Chicago Medical Center
`Chicago, Ill.
`
`Lynne S. Garcia, Editor (1997)
`UCLA Medical Center
`Los/lttgeies, Calif.
`
`Kenneth D. Thompson, Editor (1997)
`University of Chicago Medical Center
`Chicago, Iii.
`
`EDITORIAL BOARD
`
`Judith E. Domer (1993)
`Betty Ann Forbes (1995)
`
`J. Michael Miller (1994)
`Michael A. Pfaller {"1995}
`
`Daniel F. Sahm (1995)
`Steven C. Specter (1994)
`
`Linda M. Illig, Director; Jottnmls
`Barbara H. lglewslti, Chainmm, Pttblicotions Board
`J aek Kenney, Prodttcriort Editor
`
`Ciinical Microbiology Reviews considers for publication both solicited and unsolicited reviews and monographs dealing with
`all aspects of clinical microbiology. Manuscripts, proposals, and correspondence regarding editorial matters should be
`addressed to the Editor in Chief, Josephine A. Morello, Clinical Microbiology Laboratories, University of Chicago Medical
`Center, MCUU01, 5841 S. Maryland Avc., Chicago. IL 60637-1470.
`
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`Clinical
`
`Microbiology
`
`Reviews
`
`A Publication of the American Society for Microbiology
`
`VOLUMES
`
`- OCTOBEFHQQ3
`
`o NUMBEH4
`
`CONTENTS/SUMMARIES
`
`for Diagnosis of Disseminated Candidiasis.
`Nonculture Methods
`Errol Reiss and Christine J. Morrison . . . . . . . . . . . . . . . . . . . .. .
`
`311-323
`
`Sttm't?‘It2t}-‘.' Noncuiture methods to diagnose disseminated canciia'iast's (DC) are needed‘
`because biaad caitures are t1(Jtipt‘OdLtCIii—'€ for 27% or more afpaticnts with DC. Recent
`reports indicate the emergence of Candida { Torttlopsisi giabrata, Candida parapsiiosis.
`and Candida kn.tsei as agents of DC in addition to Candida aibicans and Candida
`tropicaiis. The Candida species metaboiite n—arabinitoi, expressed as serum o—arabini—
`toiicreatinine,
`is an indicator of DC in as many as two-thirds of patients studied.
`Detection is expedited by an ettzyntatic-fittorometric assay kit as an alternative to
`gas-liquid C‘h.r0t‘tm'tr)gt'aph_\-', but inteaference from niannitoi may detract from test
`specificity. Poiymerase chain reaction {PCRJ-amplified Candida species DNA has been
`recovered from blood and urine sampies front a smaii number of human subjects.
`PCR~based tests are promising but cumbersome prototypes. The sensitii-'ity to detect 1
`to 30 CFUirni of biooa’ has not been reliably achieved.
`immunoassay detection of
`marker antigens for DC has proceeded on severalfronts. A iiposomai immunoassay kit
`for the 48-kDa enoiase received a successful prospective ciinicai evaluation. Secreted
`asptntyiproteinase was cietected in urine from immunosuppressea‘ rabbits with DC. but
`data on human subjects are anai-aiiabie. Western bio: finnnttnobiot) was used to detect
`anttgenuria, and this method appears promising. The ceii wail mannoprotein {mannanj
`of Candida species circulates in the (ow naiiogram-per-miiiiiiter range in DC, but
`frequent santpiing is needed for detection during grantiiocytopenia. The incorporation
`in the santiu-ich enzyme irnrnunoassav of antibotiies of broad specificity. refiecting the
`epitopes of C. aibicans and the mannan of emerging Candida species. is necessary for
`maxintai sensiti1-‘it_v.
`
`Bacillus cereus and Related Species.
`
`Francis A. Drobniewski . . . . .
`
`324-338
`
`SItt‘tIt?Ifl'i}’.' Baciiius cereus is a gram—positi1-‘e aerobic or faculta tivety anaerobic spot'e~
`fanning rod. it is a cause offood poisoning, which isfrequentiy associatati with the
`consumption of rice-based dishes. The organism produces an emetic or diarrhea!
`.Sj,’tid.|"t}H'1t:’ ina'ttceti by an emetic toxin and enterotoxin, respecti1=c{;'. Other tarins are
`produced during growth, inciuding pnospho(ipase.s‘_. proteases. anti itemaiysins. one of
`which, cereoiysin,
`is a thioi-activated hetnoivsin. These toxins may contribute to the
`pathogenicit_v of B. cereus in nongastrointestinai disease. B. cereus isoiated from
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`
`clinicai material other than feces or vomitus was commoniy disrnisseti as a contami-
`nant, but increasingiy it is being recognized as a species with pathogenic potential. it
`is now recognized as an infrequent cause of serious nongastrointestinai in_fection,
`particniariy in drug addicts.
`the inununosuppressed, neonates, and postsurgicai pa-
`tients, especiaiiv when prosthetic impiants such as ventnicuiar shunts are inserted.
`Ocular injections are the commonest types ofse1-‘ere infection, including endophthaimi-
`tis_. panophthaimitis, and keratitis, usuaiiy with the characteristic formation ofcorneai
`ring abs'cesses. Even with prompt surgical’ and antimicrobia! agent treatment, enacte-
`ation of the eye and blindness are common sequeiae. Septicemia. meningitis. endocar-
`ditis, osteomyeiitis, and surgical and traumatic wound infections are other manifesta-
`tions ofsevere disease. B. cereus produces betu—lacttnnases, uniike Bacilius anthracis.
`and so is resistant to beta-iactam antibiotics; it is usnaily susceptible to treatment with
`clindamycin, vancomycin, gentarnicin, chioramphenicoi, and erythromicin. Simuita-
`neous therapy via muitipie routes may be required.
`
`Human Immunodeficiency Virus Type 1 Infection of the Brain. Walter
`J. Atwood, Joseph R. Berger, Richard Kaderman, Carlo S.
`Tornatore, and Eugene 0. Major . . . . . . . . . . . . .
`. .. . . . . .
`. .
`. . . . . .
`. .
`
`Summary.’ Direct infection of the centrai nervous svstetn by human immunodeficiency
`virus type I (HIV-I}, the causative agent of AIDS, was not appreciated in the early
`years of the AIDS epidemic. Neuroiogical complications associated with AIDS were
`Iargeiy attributed to opportitnistic infections that arose as a result of the immunocom-
`promised state of the patient and to depression. ln I985, set-‘era! groups succeeded in
`isolating Hi'V—i directly fi"om brain tissue. Also that year,
`the viral genome was
`cotnpietely sequenced, and HIV-I was found to beiong to a neurotropic subfamiiy of
`retrovirus known as the Lentivirinae. These findings clearfy indicated that direct HIV-1
`infection of the central nervous system played a roie in the deveiopment of/I IDS-related
`neuroiogical disease. This review summarizes the ciinical ntantfestations of HIV-1
`infection of the central nervous system and the reiated neuropathologv, the tropism of
`HR-’-I for specific ceii types both within and outside of the nervous system. the possible
`mechanisms by which HIV-Z damages the nervous system, and the current strategies
`for diagnosis and treatment ofHIV-I-associated neuropathology.
`
`John H. Rex, Michael A.
`Antifungal Susceptibility Testing.
`Pfaller, Michael G. Rinaldi, Anamarie Polak, and John N.
`Galgiani........ .
`. .
`.
`. .
`.
`. .
`. . .
`.
`.
`. . . . .
`. .
`. .
`
`367-381
`
`Sumtnary: Unlike antii;-acteriai susceptilaiiity testing, reliable uttttfitngai susceptibiiity
`testing is still iargely in its infancy. Many methods have been descn‘bed. but they
`produce widelv discrepant results uniess such factors as pH,
`inocuium size, medium
`formuiation,
`incubation time, and incubation temperature are carefitliy controlied.
`Even when iaboratories agree upon a common method, interiaboratory agreement may
`be poor. As a result of numerous coiiaborative projects carried out both independenttfv
`and under the aegis of the Subcommittee on Antifimgal Susceptibility Testing of the
`National Committee for Clinical Laboratory Standards.
`the effects of 1-'at'ying these
`factors have been extensivetj: studied and a standard method which minimizes inter-
`iaboratoty 1-'ar'iabiiit_vditt‘t'ng the testing of Candida spp. and Ctjtpt‘OC0t.‘Ctt.5‘ neoformans
`has been proposed. This review summarizes this work.
`reviews the strengths and
`weaknesses of the proposed susceptibiiity testing standard, and identifies directions for
`fitture work.
`
`Epidemiologic Evidence for Multiple Sclerosis as an Infection.
`JohnF.Kurtzke .
`. .
`. .
`.
`. .
`.
`. . . . . . . . . . . . . . . .
`
`382427
`
`Sununatg-’.‘ The worldwide distribution ofntuitipie scierosis (MS) can be described within
`three zones offrequeitcj,t' high, medium. and iow. The disease has a predilection for
`white races and for women. Migration studies show that changing residence changes
`MS risk. Studies of persons moving from high- to low-rish areas indicate that in the
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`
`high—risk areas. MS is acquired by about age I5. Minx-gas from low- to high-risk areas
`suggest that sttsceptibility is limited to persons between about ages i l and 45. M5 on
`the Fame islands has occurred as four successit-e epideniics beginning in i9-13. The
`disease appears to have been introduced by British troops who occupied the islands for
`5 yettrs from 1940, and it has remained geographically localized within the Faroes for
`half a centta}'. Whttt was introducea.’ mast have been an infection. called the primary
`MS afl’ection (PMSA). that was spread to and from successive cohorts ofFaroese. In
`this concept. PMSA is a single widespread systemic infectious disease (perhaps
`asjvmptomaticj
`that only seldom leads to clinical neurologic MS. PMSA is also
`cltaracterizecl by a need for prolonged exposure,
`limited age of sttscepttliilitjv, and
`prolonged incubation. i believe that clinical MS is the rare la te outcome of a specific,
`but unknown,
`infectious disease of aa'oiescence and young adulthood and that this
`infection could welt’ be caused by a thus-fanunidentified |‘retro)1--iras.
`
`Including the Role of the
`Infections,
`Overview of Nosocomiai
`T. Grace Emori and Robert P.
`Microbiology Laboratory.
`Gaynes...............................
`
`.S'unantajv: An estitnateti 2 million patients develop nosocomial infections in the United
`States annuallv. The increasing number ofantimtcrobial agent-resistant pathogens and
`highmislc patients in hospitals are challenges to progress in preventing and controlling
`these infections. While Esclterichia coli and Staphylococctts aurcus remain the most
`common pathogens isolated overall fivm nosocomial infections, coagttlase-negative
`staphylococci 1’CoNS), organisms prem-iouslv considered contaminants in most cultures.
`are now the predominant pathogens in bloodstream infections. The growing number of
`antimicrobial agent-resistant organisms is troabiesonie. particularly rancomycin-resis-
`tant CoNS and Entcrococcus spp. and Pseudomonas aeruginosa resistant to imipenem.
`The active involvement and cooperation ofthe microbiology lairot-atoty are important to
`the infection control program, pat-ticttlarly in sun-'eillancc and the use of laboratory
`services for epitleniiologic purposes. Surveillance is used to identify possible infection
`problems, monitor infection trends. and assess the quality of care in the hospital. it
`requires high-quaiit)-' laboratory tiara that are timely and easily accessible.
`
`LETTER TO THE EDITOR
`
`Gram-Negative Sepsis: VVhat Dilemma?
`
`James C. Hurley .
`
`. . .
`
`. . . . .
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`
`C1.lt'-IICAL MlL‘l1t'Ji3lOl_IIICu\' R.E\’IEW.'S, Oct. 1993. p. 3h?—_‘tHl
`0393-8512i'93.»"[l4l.l3h7—I5SlJ;3.[lUi[l
`Copyright ii 1'.‘i'9:’I. American Society for Microbiology
`
`This material may be protected by Copyright law (Title 17 us. Code)
`
`Vol. I5. Nu. 4
`
`Antifungal Susceptibility Testing
`JOHN H. RE)(_."° MICHAEL A. PFALLER3 MICHAEL G. RINALDLA
`ANAMARIE POLAK.‘ AND JOHN N. GALGIANI5
`
`Centerfor t'nfcr.'tiou.s‘ Diseases, Urtit-'er.rir),' of Texas Medical Schoo! at Houston. Hotisttm. Texas 1770301."
`Department ofPrzthot'og= 47}. Oregon Heahh Scicnrxs Urrit-’er:5’ir_v, Portianri. Oregon 97.Z’0I—309t5’2:
`Dcparrntenr of Pathology. Ur:iver.s‘i:y of Texas Health Science Center. Laborntor}-’ .S'enr.°'ce lU3).
`Audie L. M’m'ph_v Memorim‘ Veterans" Hospitai, San Antonio, Texas 78284-77503,"
`F. Hoflcmen-Lu Roche, Ltti, CH—4002 Basel, SH-'itzeriund",' and Median! Sen-ice 1‘ I I I l,
`I-“A Mt=dicaf C.'cmer_. and Department ofMedit1'nc,
`U.?1'il’£'.?'SiI}’ of/irizona. Tttcsuii, Arizona 857235
`
`............. "375
`
`.................... ............367
`...........................
`INTRODUCTION .............
`........................
`.................
`EARLY STUDIES ......
`FACTORS THAT INFLUENCE ANTIFUNGAL SUSCEPTIBILITY TESTING.................................369
`Endpoint Definition
`.....................
`....
`.........................
`....... ..369
`Inoculum Size ...................
`............................. ..3'l'0
`Inoculum Preparation
`.....................
`Incubation Time and Temperature............................................. .............................. ..............3'l'0
`Media
`................................
`....... "370
`Results Obtained with the Proposed NCCLS Reference Method .........
`...... ... ......... ..3'l'1
`OTHER ORGANISMS ...........
`........................
`.......................... ..372
`CORRELATION OF IN VITRO TESTING WITH IN VIVO OUTCOME
`Animal Studies
`.........................
`.....................
`Clinical Demonstrations of Both Inherent and Acquired Resistance ......
`Clinical Trials ................
`....................
`SUMMARY ..................
`..................
`Implications for the Clinical Laboratory .........
`
`........................
`.........................
`........................
`
`....................... .377
`
`INTRODUCTION
`
`infections is rising, and
`The frequency of serious fungal
`this trend has been attributed to such factors as the increas-
`ing use of cytotoxic and immunosuppressive drugs to treat
`both malignant and nonmalignant diseases,
`the increasing
`prevalence of infection due to human immunodeficiency
`virus type 1, and the widespread use of newer and more
`powerful antibacterial agents (5, 7). Fortunately,
`this in-
`crease in fungal
`infections has been accompanied by the
`development of now, less toxic. systemically active alterna-
`tives to amphotericin B such as fluconazole, itraconazolc,
`and the various amphotcricin B lipid formulations (129).
`With this proliferation of antifungal agents,
`therapeutic
`options are more numerous. and the clinician must now
`choose among them. This decision is made more difficult by
`the steady stream of reports of putative drug resistance to
`one or more antifungal agents (6. 13, 16, 26, 32, 33, 38, 39,
`46, 47, 62, 69, 70, 72, 75. 89, 93, 103. 105. 119, 120, 126. 136,
`138, 146, 150-152). As these reports are not always Convinc-
`ing or well controlled. making an informed antifungal ther-
`apy choice is not easy.
`As with bacterial infections. the clinician would like to be
`guided by knowledge of local epidemiological patterns de-
`rived from drug susceptibility testing results. Unlike antibac-
`terial susccptibility testing, however, reliable antifungal sus-
`ceptibility testing is still largely in its infancy. A prodigious
`array of techniques has been described. but without stan-
`
`’ Corresponding author.
`
`3:37
`
`the various methods; have produced widely
`dardization.
`discrepant results because of substantial dependence on
`such factors as pH, inoculum size. liquid versus solid media,
`medium formulation, time ol' incubation, and temperature of
`incubation. Even when a group of laboratories agrees upon a
`general
`technique. widcly discrepant results may be ob-
`tained for some antifungal agents if all procedural details are
`not precisely defined {I9}. Finally, once :1
`technique is
`selected and the MIC for an isolate is determined. there are
`few useful data available to guide MIC interpretation. Many
`authors have sought in vivo-in vitro correlations in animal
`models or with isolates from patients: some have found a
`correlation [3, 14. 39. ‘I2, 85, 93, 104, 105, 119, 120, 122. 127,
`128, I36, 139, 149). but others have not (20. 47. S5. 73, 86,
`127, 134). This situation is obviously unsatisfactory. and a
`substantial amount of work has been undertaken to resolve
`
`these difficulties. While a complete solution is not available,
`considerable progress has been made, and it is the purpose
`of this review to bring this information into perspective.
`
`EARLY STUDIES
`
`Antifungal susceptibility testing was not relevant until the
`introduction in the 19505 of arnphotcricin 13. That this was
`nearly 30 years after the discovery of the first antibacterial
`agents explains in part the immaturity of antifungal suscep-
`tibility tcsting today. Even then. antilungal susceptibility
`testing lay fallow for many years because, while not all
`fungal infections responded to amphotcricin B, there were
`no alternatives. It was only with the dcvcloprnc-nt of S-fluo-
`rocytosinc and, more recently. the uzole antifungal agents
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`368
`
`REX ET A]...
`
`CLIN. MlC‘ROBlOL.. Rev.
`
`that differences within and between species started to be-
`come. apparent. Even so, only a small number of laboratories
`routinely performed antifungal susceptibility testing.
`In
`1936, Calhoun et al. (19) surveyed 350 hospital laboratories.
`Only 41 of the 210 respondents were performing antifungal
`susceptibility tests, with 45% of these laboratories testing
`only one to five isolates during the previous year. A variety
`of methods was being used: most commonly, a broth tech-
`nique that had been derived from an antibacterial suscepti-
`bility testing method. When seven laboratories used this
`common, published methodology to test five. Candida albi-
`etms isolates against amphotericin B and 5-tluorocytosine,
`results that varied by as much as 512-fold were obtained- Of
`significant interest, however, was the observation that while
`the absolute values of the MICS for each isolate varied
`greatly among laboratories,
`the relative susceptibilities of
`the isolates varied little "among laboratories.
`In other
`words, the rank order of the isolates was quite constant
`despite wide. variability in endpoint results. in a follow-up
`study conducted by three laboratories (S1}, another set of
`Cortdida isolates was tested against amphotericin B, 5-fluo-
`rocytosinc, and ltetoeonazole.
`In this study, however. no
`methodology was prescribed. Rather. each group used the
`method with which they were most familiar, and the imple-
`mented techniques included broth dilution, agar dilution,
`and turbidirnetric techniques. Again, while the actual MICS
`varied by as much as 50,000-fold,
`the rank order of the
`isolates produced by the results from each laboratory varied
`only slightly-
`Given these data and the growing interest in antifungal
`susceptibility testing, it was clear that standard. reproduc-
`ible techniques for antifungal susceptibility testing were
`needed. Many approaches to antifungal susceptibility testing
`have been reported, but not all readily lend themselves to
`standardization. Tests that measure the rate of elongation of
`germ tubes (75. 136, 151] are tedious to perform and are
`useful only for C. albicans, the one species in the genus that
`forms germ tubes within 3 h in serum. An automated system
`based on the dynamic growth of a single hypha as measured
`by microscope, camera, television monitor, video tape re-
`corder. and microcomputer was recently described (102,
`154), but it appears impractical for routine use. Examination
`of even more subtle morphologic eifccts is possible and
`would be applicable to a wider range of fungi (121, 136), but
`such approaches are highly subjective. Measurements of
`uptake of radiolabeled metabolites (30, 31, 35, 56} or reduc-
`tion of colored substrates (79, 124) have been used to assess
`phagocyte-mediated damage to C. rztbicans, Aspergitlns fit-
`migartts, RJ'1..fZOp£tS ttrrhiztts (_=R. oiyzae), and Cocrridioides
`immiris and could readily be adapted to measuring the effect
`of antifungal drugs on fungi (88, 140. 143). However. these
`tests are indirect since lack of metabolic uptake of the given
`substrate does not necessarily imply death. Moreover, they
`require special material-handling procedures in the case of
`the use of radiolabeled substrates and relatively large inocuia
`in order to produce a measurable amount of product in the
`case of colored substrates. Approaches that use flow cytoto-
`etry (60, 116-118") or viable colony counts (71, 95) have bec.n
`described but are applicable only to organisms that disperse
`freely and are technically or physically demanding. Mea-
`surement of biomass by bioltuninescence spectrophotometry
`has been described (95). This technique used nteasurernents
`at multiple points along a dose-response curve.
`to allow
`computation of a relative inhibition factor (100), and al-
`though the method is intriguing. instrumentation that would
`
`make this approach practical has not been developed fur-
`ther.
`
`Agar-based techniques have been used extensively by a
`few laboratories because they are simple, economical. and
`easy to perform simultaneously on large numbers of organ-
`isms. Either the. fungus can be placed in the agar and the
`antifungal agent can be placed on the surface of the agar
`(disk or well diffusion) or vice versa [agar dilution). These
`techniques suffer from substantial dependence on inoculurn.
`temperature. and duration of incubation (15, 66, 67, 75. 94').
`The estimation of the size of inhibitory zones can be difficult
`because of partial growth inhibition (3't', 6?, 139'] or the
`presence of “persistor” [sic] colonies within otherwise clear
`zones of inhibition (145). Finally, the absolute MICs of the
`azoles tend to be lower than those produced by broth assays
`(15, 94}. Some of these problems might be overcome by
`careful standardization (59), but the physical-chemical prop-
`erties of the antifungal agents to be tested and their interac-
`tion with the agar cannot be altered. Agar is not a chemically
`defined material, and, as has been noted in assays of the
`aminocyclitol antibiotics, agars from different sources can
`produce different results because of variations in such simple
`properties as cation concentration (66). Such variations are
`also potentially relevant to antifungal assays (94). In addi-
`tion, some (61, 68), but not all (130). authors have found that
`amphotericin B and the azoles tend to deteriorate when
`stored in dilute form or dried on antibiotic assay disks. Some
`of the azolcs. especially miconazole,
`l-(etoconazole, and
`itraconazole. are relatively insoluble and may diffuse poorly
`in an agar diffusion system (61,
`I31, 129]. Finally, some
`species ofCandt'dn other than C. afbicaits are inhibited when
`grown in agar (W). Despite these problems,
`it has been
`possible to develop routine testing methods that use an agar
`diffusion format [133]. Reproducible results that correlate
`well with broth dilution MICs have been obtained (37. 115,
`130), and standard techniques for performing this assay have
`been described (111, 137}.
`In addition, many reports of
`amphotericin B resistance have used agar dilution methods
`(:32, 33, 62, 8?, 89, 103). Whether currently proposed broth
`l'!‘lEIl'lCI(.lS can detect this resistance needs to be determined
`(see reference 50 and below).
`Broth dilution methods for antifungal susceptibility testing
`were t_he most commonly used techniques in the United
`States in the previously mentioned survey (19). Because of
`this and the other factors just discussed, broth dilution
`methods have been the focus of the most recent efforts at
`test standardization. A number of collaborative studies of
`broth dilution methods have now been carried out both
`
`independently (43, 63, 111, 135, 144) and under the aegis of
`the Subcommittee on Antifungal Susceptibility Testing of
`the National Committee for Clinical Laboratory Standards
`(NCCLSJ (19, 44, -18. S1, 107, 108). As a result, many of the
`factors that influence {often profoundly) this technique have
`been identified, and a standard method that minimizes their
`adverse effects has been proposed {Q2}. This method has
`been proposed exclusively for testing Candida spp. and
`Ciyptococcus ueoformtzn.-t; reproducible methods for other
`organisms are still under development (see below). In the
`next section. we review the variables involved in implement-
`ing a broth-based susceptibility test, with special emphasis
`on how each variable affects the proposed NCCLS reference
`method (Table 1). Knowledge of these variables is helpful in
`understanding the relative importance of the various details
`of the assay and in interpreting studies that use variant
`methodologies.
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`ANTIFLTNGAI. SUSCEPTIBILITY TESTING
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`FIG. 1. Trailing endpoints. A C. nlbicmis isolate was tested for
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`percent transmission at 53lJ nm was determined. The lines marked
`80 and 90% are the percent transmission of 1:5 and 1:10 dilutions of
`the drug-free control tube, respectively. The amphotcricin 3 curve
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`FACTORS THAT INFLUENCE ANTIFUNGAL
`SU SCEPTIBILITY TESTING
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`Endpoint Definition
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`Endpoint determination is probably the most significant
`source of interlaboratory variability for the azole antifungal
`agents and, under some conditions, 5-fluorocytosinc. For
`these agents, inhibition does not develop abruptly over a
`small concentration range. Rather, after an initial prominent
`drug effect, small amounts of turbidity may persist for many,
`if not all, drug concentrations tested (Fig. 1)- As a result,
`laboratories that insist on stringent reduction of turbidity
`report much higher MICs than do laboratories that adopt
`endpoints that are tolerant of small amounts of turbidity.
`In contrast, endpoints with amphotcricin B and cilofungin
`do not show this phenomenon, and comparison of the
`kinetics of the onset of drug efiect has provided some
`explanation for diflerenccs between these drugs [54, 85}. At
`or above some critical amphotcricin B concentration. turbi-
`dimetric measurements demonstrate that growth ceases
`soon alter exposure to the drug. However. growth does not
`begin to slow until approximately one doubling time after
`cxposurc to 5-fluorocytosine or the azole antifungai agents,
`and growth is not fully arrested until some time later. These
`observations demonstrate that this trailing endpoint is the
`result of growth occurring for a period of time prior to the
`onset of complete drug eifcct. Predictably. the trailing end-
`point problem has been shown to worsen with increasing
`initial inoculum (127), and this may be the reason that recent
`collaborative trials have found better reproducibility with a
`smaller inoculum (27. 44, 48). It has been suggested that this
`problem could be reduced by the addition to the medium of
`a protein synthesis inhibitor such as doxycycline (96, 101'}.
`but this approach has not been exploited further.
`Given these observations, several approaches to endpoint
`definition are possible. One is to describe the endpoint with
`carefully selected phrases. This was done in two recent
`collaborative studies in which endpoint determinations of
`“optically clear." “slightly hazy,” and “prominent de-
`crease in turbidity from control" were used (44. 48]. (These
`descriptions are also referred to as 0, 1+, and 2+, respec-
`tively.) It was found that the endpoints that allowed some
`
`CFAD V. Anacor, |PR2015-01776 ANACOR EX. 2108 - 8/20
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`
`370
`
`REX ET AL.
`
`CLI1-.1- MICROBIOL. Rev.
`
`turbidity gave more reproducible results and also produced
`better agreement with known patterns of in vivo resistance.
`While some trade-offs were involved, the “prominent de-
`crease in turbidity from control” endpoint was the most
`consistently reproducible.
`Communicating the precise meaning of "prominent de-
`crease in turbidity from control" is, however, problematic.
`An approach to the resolution of this problem has been
`described by Espinel-lngrotl“ et al. (44). These authors d