Comparative ofBroth Macrodilution and Microdilution Techniques … · Malattie Infettive e Medicina...

JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1994, p. 2494-2500 Vol. 32, No. 100095-1137/94/$04.00+0Copyright © 1994, American Society for Microbiology

Comparative Study of Broth Macrodilution and MicrodilutionTechniques for In Vitro Antifungal Susceptibility Testing of

Yeasts by Using the National Committee for ClinicalLaboratory Standards' Proposed Standard

FRANCESCO BARCHIESI,1,2* ARNALDO L. COLOMBO,'3 DEANNA A. McGOUGH,'AND MICHAEL G. RINALDI"14

Fungus Testing Laboratory, Department of Pathology, University of Texas Health Science Center, San Antonio, Texas78284-77501, Audie L. Murphy Memorial Veterans Hospital, San Antonio, Texas 78284-51004; Istituto di

Malattie Infettive e Medicina Pubblica, Universitai degli Studi di Ancona, Ancona, Italy2,and Escola Paulista de Medicina, Sao Paulo, Brazil3

Received 7 April 1994/Returned for modification 11 June 1994/Accepted 2 July 1994

A comparative study of broth macro- and microdilution methods for susceptibility testing of fluconazole,itraconazole, flucytosine, and amphotericin B was conducted with 273 yeasts. The clinical isolates included 100Candida albicans, 28 Candida tropicalis, 25 Candida parapsilosis, 15 Candida lusitaniae, 15 Candida krusei, 50Cryptococcus neoformans var. neoformans, 25 Torulopsis (Candida) glabrata, and 15 Trichosporon beigelii strains.Both methods were performed according to the National Committee for Clinical Laboratory Standards'(NCCLS) recommendations (document M27-P). For fluconazole, itraconazole, and flucytosine, the endpointwas the tube that showed 80% growth inhibition compared with the growth control for the macrodilutionmethod and the well with slightly hazy turbidity (score 1) compared with the growth control for themicrodilution method. For amphotericin B, the endpoint was the tube and/or well in which there was absenceof growth. For the reference macrodilution method, the MICs were determined after 48 h of incubation forCandida spp., T. glabrata, and T. beigeii and after 72 h for C. neoformans var. neoformans. For the microdilutionmethod, either the first-day MICs (24 h for all isolates other than C. neoformans var. neoformans and 48 h forC. neoformans var. neoformans) or the second-day MICs (48 and 72 h, respectively) were evaluated. Theagreement within one doubling dilution of the macrodilution reference for all drugs was higher with thesecond-day MICs than with the first-day MICs for the microdilution test for most of the tested strains. Generalagreement was 92% for fluconazole, 85.7% for itraconazole, 98.3% for flucytosine, and 96.4% for amphotericinB. For C. neoformans var. neoformans and T. beigelii, the agreement of the first-day reading was higher than thatof the second-day reading for fluconazole (94 versus 92%, respectively, for C. neoformans var. neoformans, and86.7 versus 80%, respectively, for T. beigelii). Our studies indicate that the microdilution technique performedfollowing the NCCLS guidelines with a second-day reading is a valid alternative method for testing fluconazole,itraconazole, flucytosine, and amphotericin B against these eight species of yeasts.

Infections caused by fungi have become more frequent inpart because of improved diagnosis and the increasing numberof immunocompromised patients (4). At the same time, sev-eral new antifungal agents have become available, resulting inmore therapeutic options and a greater demand for in vitrosusceptibility testing (6). These tests, like those for antibacte-rial agents, may be an important aid in the initiation andmonitoring of antifungal therapy. Susceptibility testing withantifungal agents, however, is greatly influenced by a variety offactors, including inoculum size, strain morphology, physicaland chemical properties of the antifungal agent being tested,medium used for testing, and temperature and duration ofincubation (8, 9, 14-16, 22, 24, 25, 27, 30, 31, 33, 34). Inaddition, one of the major sources of susceptibility test varia-tion for antifungal agents, especially for azoles, is the phenom-enon of partial inhibition of fungal growth, or trailing, whichmakes endpoint determination difficult (2, 11, 13, 17, 28, 30,33-35).

* Corresponding author. Mailing address: Fungus Testing Labora-tory, Department of Pathology, University of Texas Health ScienceCenter at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX78284-7750. Phone: (210) 567-4131. Fax: (210) 567-4076.

After several collaborative studies, the Subcommittee onAntifungal Susceptibility Tests of the National Committee forClinical Laboratory Standard (NCCLS) published a proposedstandard for the broth dilution antifungal susceptibility testingof yeasts (23). A broth macrodilution technique performed inRPMI 1640 buffered at pH 7.0 with MOPS (morpholinepro-panesulfonic acid), a spectrophotometrically standardized in-oculum, incubation at 35°C, and second-day reading (48 h forCandida spp. and Torulopsis [Candida] glabrata and 72 h forCryptococcus neoformans var. neoformans) were the parame-ters chosen by the NCCLS subcommittee for standardizing thereference method (23). Broth microdilution antifungal suscep-tibility testing is similar to broth macrodilution testing, butsmaller amounts of medium, reagents, and antifungal agentsare needed (2, 10, 11, 18, 28). Thus far, the few studies thathave been conducted to compare macro- and microdilutionantifungal susceptibility testing of yeasts have shown a goodagreement between macro- and microdilution methods fortesting amphotericin B, flucytosine, ketoconazole, and flucon-azole when the recommendations provided by the NCCLS forthe macrodilution tests were applied for both methods (10, 11,28).

In this study, we compared macro- and microdilution tech-

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niques by determining the MICs of four antifungal agentsagainst 273 clinical yeast isolates. The purpose of the presentinvestigation was to (i) provide additional data comparingmicrodilution testing of fluconazole, flucytosine, and ampho-tericin B with the reference macrodilution method and (ii)provide new data comparing micro- and macrodilution testingof itraconazole using the test conditions in keeping with theNCCLS guidelines.

MATERIALS AND METHODS

Antifungal agents. The following four antifungal agentswere used: fluconazole (Pfizer, Inc., New York, N.Y.), itracon-azole (Janssen Pharmaceutica, Titusville, N.J.), flucytosine(Hoffmann-La Roche Laboratories, Inc., Nutley, N.J.), andamphotericin B (E. R. Squibb & Sons, Princeton, N.J.). Thefour drugs were provided by the manufacturers as standardpowders, and stock solutions were prepared with the weightadjusted according to the potency of each drug. Stock solutionsof fluconazole and flucytosine, each at 16,000 ,ug/ml, wereprepared with sterile distilled water. A stock solution ofitraconazole, at 5,000 ,ug/ml, was prepared in polyethyleneglycol 400 (Union Carbide, Danbury, Conn.), with the aid ofheating at 75°C for 45 min. Amphotericin B was dissolved in100% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.)to obtain a stock solution of 16,000 ,ug/ml.

Test organisms. A panel of 273 clinical isolates of patho-genic yeasts were used in this study. The clinical isolatesincluded 100 isolates of Candida albicans, 28 isolates ofCandida tropicalis, 25 isolates each of Candida parapsilosis andTorulopsis (Candida) glabrata, 15 isolates each of Candidalusitaniae, Candida krusei, and Trichosporon beigelii, and 50isolates of Cryptococcus neoformans var. neofonnans. Eachisolate represented a unique isolate from a patient. All yeastisolates were identified to the species level by the API 20Csystem (Analytab Products, Plainview, N.Y.), supplemented asneeded with conventional morphologic and biochemical meth-ods, and maintained as water suspensions at room temperature(38). Canavanine-glycine-bromothymol blue agar was used toidentify the C. neoformans var. neofornans isolates (21). Twoadditional isolates, C. albicans ATCC 90029 and C. krusei93-1380 (an isolate from the collection of the Fungus TestingLaboratory, Department of Pathology, University of TexasHealth Science Center at San Antonio), were included in thisstudy as reference control organisms and were tested by bothmethods in each series of assays.

Susceptibility testing procedure. Each isolate was simulta-neously tested by the reference macrodilution method and bya microdilution method. The recommendations provided bythe NCCLS subcommittee for broth macrodilution susceptibil-ity testing procedure were used for both techniques (23).

(i) Macrodilution method. Susceptibility testing was per-formed in RPMI 1640 medium (American Biorganics, Inc.,Niagara Falls, N.Y.), with L-glutamine and without sodiumbicarbonate, and buffered at pH 7.0 with 0.165 M morpho-linepropanesulfonic acid (MOPS). Drug dilutions were pre-pared at 10 times the strength of the final drug concentrationby additive drug dilution schemes for minimizing systematicpipetting errors. Drug dilutions were stored at -70°C untilused. The yeast isolates were grown on Sabouraud dextroseagar for 24 to 48 h at 35°C and subcultured twice to ensuretheir purity and viability. The inoculum suspension was pre-pared by picking five colonies of at least 1 mm in diameter andsuspending them in 5 ml of sterile 0.85% saline. The resultingsuspension was vortexed for 15 s, and the cell density wasadjusted with a spectrophotometer by adding sufficient sterile

saline to increase the transmittance to that produced by a 0.5McFarland standard at a 530-nm wavelength. The workingsuspension was made by a 1:100 dilution followed by a 1:20dilution in RPMI 1640 broth medium in sufficient volume todirectly inoculate each MIC tube with 0.9 ml. Inoculum sizeswere confirmed with the final higher inoculum for all thestrains tested by enumeration of the CFU per milliliter onsubcultures on Sabouraud dextrose agar. Yeast inocula (0.9ml) were added to the 1Ox drug dilutions in polystyrene plastictubes (12 by 75 mm; Falcon 2054; Becton Dickinson, LincolnPark, N.J.), bringing the drug dilutions to the final testconcentrations: 0.125 to 64 ,ug/ml for fluconazole and flucy-tosine and 0.03 to 16 ,ug/ml for itraconazole and amphotericinB. Drug-free and yeast control tubes were included for eachisolate tested. All tubes were incubated without agitation at35°C and read at 48 h for all the isolates except C. neoformansvar. neoformans, which was read at 72 h. Before reading, eachtube was flicked, and its turbidity was compared with that ofthe growth control (drug-free) tube. Fluconazole, itraconazole,and flucytosine MICs were defined as the lowest drug concen-tration which resulted in a visual turbidity less than or equal to80% inhibition compared with that produced by the growthcontrol (0.2 ml of growth control plus 0.8 ml of uninoculatedRPMI 1640). Amphotericin B MICs were defined as the lowestdrug concentration at which there was an absence of growth.

(ii) Microdilution method. Broth microdilution testing wasperformed in sterile, flat-bottomed 96-well microplates (Fal-con 3072; Becton Dickinson). The 1OX drug dilutions werediluted 1:5 with RPMI 1640 to obtain 2 times the finalconcentrations. Volumes of 100 ,lI of the 2x drug dilutionswere dispensed into the wells with a multichannel pipette(Digital Multichannel Pipette; Labsystems, Helsinki, Finland)with wells 1 and 10 of each row containing the highest and thelowest concentrations, respectively. Wells 11 and 12 of eachrow served as the growth control and sterility check, respec-tively. The microplates were stored at -70°C until used. Theworking suspension of the inoculum, prepared spectrophoto-metrically as described above, was made by a 1:50 dilutionfollowed by a 1:20 dilution in RPMI 1640 to obtain a 2x finalsuspension.Inoculum sizes were also confirmed for the microdilution

test with the final higher 2x suspension for all the strainstested by the enumeration of the CFU per milliliter onsubcultures on Sabouraud dextrose agar. One hundred micro-liters of the yeast suspension was added to each well, resultingin the desired final drug concentration and inoculum size. Themicroplates were incubated at 35°C and read at 24 and 48 h(first- and second-day readings) for all the isolates except C.neofornans var. neoformans, which was read at 48 and 72 h(first- and second-day reading). Before reading, microplatescontaining fluconazole, itraconazole, and flucytosine were ag-itated for 5 min by a microtiter plate shaker (DynatechLaboratories, Inc., Chantilly, Va.). In order to detect theminimum amount of growth needed to define the amphoteri-cin B MICs, microplates containing this drug were readwithout agitation. The microdilution wells were scored with theaid of a reading mirror. The growth in each well was comparedwith that of the growth control (drug-free) well. A numericalscore was given to each well by using the following scale: 0,optically clear; 1, slightly hazy; 2, prominent decrease inturbidity (approximately 50% inhibition); 3, slight reduction inturbidity; and 4, no reduction in turbidity. Fluconazole, itra-conazole, and flucytosine MICs were defined as the lowestdrug concentration which resulted in a slightly hazy turbidity(score 1) compared with that in the growth control (drug-free)

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TABLE 1. Macrodilution and microdilution antifungal susceptibilities of fluconazole against 275 yeasts

Fluconazole MIC (pLg/ml)a

Fungus (no. tested) Macro Micro (1st reading) Micro (2nd reading)

Range MIC50 MIC90 Range MIC50 MICgo Range MIC50 MICgo

C. albicans (100) 0.25->64 4 >64 .0.125->64 4 64 0.25->64 4 >64C. tropicalis (28) 0.5->64 1 64 0.125-16 0.5 1 0.25->64 1 64C. parapsilosis (25) 0.5-4 1 4 0.25-2 0.5 1 0.25-4 1 2C. lusitaniae (15) 0.25-32 1 32 0.25-32 0.5 2 0.25-32 1 32C. krusei (15) 32-64 64 64 8-32 16 32 32->64 64 >64C. neoformans (50) 0.5->64 4 16 0.25-64 4 16 0.5-64 4 16T. glabrata (25) 1->64 16 >64 0.25->64 4 32 1->64 8 >64T. beigelii (15) 1-64 4 8 0.25-32 2 4 2-64 4 16C. albicans ATCC 90029" 0.5 0.25-0.5 0.5C. krusei 93-1380b 64 32 >64

a For the reference broth macrodilution method (macro), the tubes were read after 48 h of incubation for all the isolates except C. neoformans, which was read at72 h (23). For the microdilution method (micro) either the first-day reading (24 h for all isolates other than C. neofornans and 48 h for C. neoformans) or the second-dayreading (48 h and 72 h, respectively) was evaluated.

b Each reference strain was tested in 27 separate sets of experiments by both methods.

well. The amphotericin B MIC was defined as the lowest drugconcentration at which there was an absence of growth.

Analysis of the results. All the microdilution first- andsecond-day reading MICs were compared with reference ma-

crodilution MICs. Both on-scale and off-scale results were

included in the analysis. The high off-scale MICs (>64 and>16 p.g/ml) were converted to the next highest concentrations(128 and 32 jig/ml), and the low off-scale MICs (.0.125 andc0.03 pg/ml) were left unchanged. When skips (uneven pat-terns) were present, the MIC endpoint was the higher drugconcentration. Differences among MIC endpoints of no more

than one dilution (one tube or well) were used to calculate thepercent agreement.

RESULTS

A total of 3,924 test results were obtained in this study by 27different sets of experiments. All the organisms tested pro-duced detectable growth after 48 h (72 h for C. neoformans var.

neoformans) of incubation by the reference macrodilutionmethod for the four drugs. One isolate of T glabrata failed toproduce detectable growth on the first day's reading in themicroplates for all four drugs. Twenty-seven isolates (11isolates of T. beigelii, 7 isolates of C. neoformans var. neofor-mans, 4 isolates each of C. parapsilosis and T. glabrata, and 1isolate of C. lusitaniae) failed to produce detectable growth at

the first day's reading in microplates for itraconazole. All thetested organisms produced detectable growth at the secondday's reading in microplates with the exception of one isolateof C. neoformans var. neoformans. Therefore, there was inad-equate growth in a total of 32 test results by the microdilutionmethod (31 at the first reading and one at the second reading).These data, however, while considered in disagreement withthe reference macrodilution results, were nevertheless in-cluded in the statistical analysis. Despite the large variability inthe sizes of the yeast cells among different species and withinthe same species, the spectrophotometric preparation of theinoculum resulted in the same log phase (103) for all theorganisms tested by both methods.The MIC ranges and the MICs required to inhibit 50 and

90% of the isolates (MIC50s and MIC90s, respectively) of thefour drugs are summarized in Tables 1 to 4. Fluconazole MICsshowed a broad range for all the species with the exception ofC. parapsilosis and C. krusei (Table 1). Itraconazole MICsshowed a broad range only for C. albicans (Table 2). This was

because one isolate of C. albicans was highly resistant to thistriazole. Flucytosine MICs showed a broad range for all thespecies with the exception of C. krusei and T. beigelii (Table 3).Amphotericin B showed a very narrow range for all the speciestested (Table 4). Generally, the MIC50s and MIC90s of the fourdrugs obtained by the second reading of the microdilution testwere more comparable with the reference macrodilution re-

TABLE 2. Macrodilution and microdilution antifungal susceptibilities of itraconazole against 275 yeastsa

Itraconazole MIC (,ug/ml)


Range MIC50 MICg< Range MIC50 MICgo Range M'C50 MIC90

C. albicans (100) .0.03->16 0.125 1 <0.03->16 0.06 1 .0.03->16 0.125 1C. tropicalis (28) 0.125-1 0.125 0.5 .0.03-0.5 0.03 0.5 0.03-0.5 0.125 0.5C. parapsilosis (25) .0.03-0.25 0.06 0.125 .0.03-0.5 0.125 0.125 0.03-0.5 0.125 0.5C. lusitaniae (15) .0.03-0.25 0.06 0.25 .0.03-0.125 s0.03 0.06 .0.03-0.5 0.06 0.25C. krusei (15) 0.125-0.5 0.25 0.5 0.125-0.5 0.125 0.125 0.25-0.5 0.5 0.5C. neoformans (50) .0.03-0.5 0.125 0.25 -0.03-0.5 0.125 0.5 0.03-1 0.125 0.5T. glabrata (25) .0.03-4 0.5 2 .0.03-0.5 .0.03 0.5 .0.03-1 0.5 1T. beigelii (15) .0.03-0.25 0.06 0.25 <0.03-0.125 .0.03 0.125 .0.03-0.5 .0.03 0.5C. albicans ATCC 90029 0.03 0.03 .0.03C. krusei 93-1380 0.25 0.5 0.5

a See Table 1, footnotes a and b.

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TABLE 3. Macrodilution and microdilution antifungal susceptibilities of flucytosine against 275 yeasts'

Flucytosine MIC (,ug/ml)


Range MIC50 MIC9o Range MIC50 MICgJ Range MIC50 MICgoC. albicans (100) <0.125->64 0.5 4 <0.125->64 <0.125 1 <0.125->64 0.25 8C. tropicalis (28) '0.125->64 0.25 1 <0.125-32 <0.125 0.25 <0.125->64 0.25 1C. parapsiosis (25) '0.125->64 '0.125 0.5 '0.125-1 .0.125 .0.125 s0.125->64 .0.125 <0.125C. lusitaniae (15) <0.125->64 <0.125 >64 '0.125->64 '0.125 16 <0.125->64 s0.125 >64C. krusei (15) 4-16 8 16 4-8 4 8 8-16 8 16C. neoformans (50) 1->64 4 8 0.5-64 2 8 1->64 4 8T. glabrata (25) '0.125->64 .0.125 '0.125 <0.125->64 '0.125 <0.125 s0.125->64 <0.125 <0.125T. beigelii (15) 8->64 32 >64 1-64 8 64 8->64 16 >64C. albicans ATCC 90029 >64 .64 >64C. kusei 93-1380 8 8 8


sults than those obtained by the first reading for all the species azole, itraconazole, flucytosine, and amphotericin B being 92,(Tables 1 to 4). 85.7, 98.3 and 96.4%, respectively (Table 6).The percentage of agreement between the macro- and

microdilution methods for the four drugs is shown in Table 5. DISCUSSIONAs expected by the analysis of MIC50s and MIC90s, theagreement was higher at the second- than the first-day reading In this study, we compared the MICs of fluconazole, itra-in the microdilution test. In contrast, however, there was conazole, flucytosine, and amphotericin B by broth macro- andgreater agreement with C. neoformans var. neoformans and T. microdilution techniques against eight different species ofbeigelii tested against fluconazole at the first day's reading yeasts. The recommendations provided by the NCCLS for the(Table 5). The largest discrepancies between the first and macrodilution method were also used in the microdilutionsecond readings were found when testing fluconazole against procedure. Although the in vitro method for susceptibilityC. tropicalis, C. parapsilosis, C. krusei, and T. glabrata; itracon- testing of yeasts reported by the NCCLS document has beenazole against C. tropicalis, C. lusitaniae, C. krusei, and T. proposed for only flucytosine, amphotericin B, ketoconazole,glabrata; flucytosine against C. albicans, C. tropicalis, and T. and fluconazole, we applied these same parameters for testingbeigelii; and amphotericin B against C. parapsilosis and T. itraconazole as well. Our data showed that the microtiterbeigelii (Table 5). The number of differences for second-day technique, which is both more economical and less cumber-readings of microdilution tests was significantly lower than that some than the macrodilution test, is an adequate tool forobtained in the first-day reading for the four drugs against all antifungal susceptibility testing when performed by the recom-species (P = 0.002, Student's t test). The only isolates showing mendations provided by the NCCLS for the proposed mac-no differences between the first and second readings in the rodilution standard. It also corroborated data reported bymicrodilution and reference macrodilution methods were C. Espinel-Ingroff et al. indicating excellent agreement betweenkrusei and T. glabrata for flucytosine and C. lusitaniae for macro- and microdilution methods when testing fluconazoleamphotericin B (Table 6). The values obtained with the (92%), flucytosine (98.3%), and amphotericin B (96.4%)reference macrodilution method tended to be higher than against a large number of clinical isolates of yeasts (11).those obtained by first reading of the microdilution method for Recently, Pfaller et al., comparing macro- and microdilutionthe four drugs against all the species tested. The total agree- susceptibility testing of fluconazole against 119 clinical isolatesment between macro- and second-day readings of microdilu- of C. albicans, found 94 and 93% agreement at 24 and 48 h,tion tests was generally excellent, with the values for flucon- respectively (28). In our study, we found 97% agreement

TABLE 4. Macrodilution and microdilution antifungal susceptibilities of amphotericin B against 275 yeastsa

Amphotericin B MIC (,g/ml)

Fungus (no. tested) Macro Micro (1st reading) Micro (2nd reading)Range MIC50 MICgo Range MIC50 MIC,o Range MIC50 MICgo

C. albicans (100) 0.25-2 0.5 1 0.25-1 0.5 0.5 0.25-1 0.5 0.5C. tropicalis (28) 0.5-1 1 1 0.25-2 1 2 0.5-2 1 2C. parapsilosis (25) 0.5-2 1 2 .0.03-0.5 0.25 0.5 0.5-1 1 1C. lusitaniae (15) 0.5-1 0.5 1 0.25-1 0.5 1 0.25-1 1 1C. krusei (15) 1-2 1 2 0.25-1 1 1 0.5-2 1 2C. neoformans (50) 0.25-1 0.5 1 0.06-1 0.25 0.5 0.125-1 0.5 0.5T. glabrata (25) 0.5-1 0.5 1 0.06-1 0.25 0.5 0.25-1 0.5 1T. beigelii (15) 0.5-2 1 2 0.25-2 0.5 1 0.25-2 2 2C. albicans ATCC 90029 0.5 0.25-0.5 0.5-1C. krusei 93-1380 1 0.5 1


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TABLE 5. Agreement between macro- and microdilution methods for four drugs tested against eight different species of yeasts

% Agreement between macrodilution results and 1st and 2nd microdilution results"Fungus (no. tested)

Fluconazole Itraconazole Flucytosine Amphotericin B

C. albicans (100) 84/97 74/75 64/96 94/98C. tropicalis (28) 50/85.7 32.1/75 53.5/96.4 92.8/96.4C. parapsilosis (25) 52/88 56/72 88/96 64/96C. lusitaniae (15) 80/93.3 66.6/100 93.3/100 100/100C. krusei (15) 60/100 60/100 100/100 86.6/100C. neoformnans (50) 94/92 78/82 96/98 88/92T. glabrata (25) 28/100 28/88 100/100 84/96T. beigelii (15) 86.7/80 26.7/93.3 26.7/100 73.3/93.3

a Values are shown as first reading/second reading.

between the reference macrodilution and microdilution flu-conazole susceptibility tests read at 48 h for 100 isolates of C.albicans. Our agreement at 24 h was lower (84%). Moreover,we found that the second-day reading of the microtiter platesgave results consistently closer to those obtained by thereference macrodilution test for the four drugs against all thespecies tested. This could be of clinical relevance, since theresults reported at the first-day reading might suggest falsesusceptibilities of the isolates tested, mainly for flucytosine andthe azole drugs.Our results displayed a dramatic increase in the percent

agreement from 24 to 48 h when testing fluconazole (28 versus100%, respectively) and itraconazole (28 versus 88%, respec-tively) against T. glabrata, a species often reported to beresistant to the azoles in vitro as well as in vivo (25, 37). Anincrease in agreement from 24 to 48 h was also found whenboth azoles were tested against C. krusei, another speciesdemonstrating resistance to the azoles (1, 25, 40). For flucy-tosine, a large discrepancy between the first- and second-dayreadings was found for isolates of T beigelii. In vitro, the T.beigelii isolates appeared to be less susceptible when tested bythe reference macrodilution and by the second-day reading ofthe microdilution method (MIC range, 8 to >64 ,ug/ml). Fourisolates of C. tropicalis and one isolate each of C. albicans andC. lusitaniae showing in vitro susceptibility to fluconazole in themicrodilution test at 24 h (MIC range, 0.25 to 1 ,ug/ml)appeared to be less susceptible to this triazole by the referencemacrodilution and by the 48-h reading with the microdilutiontests (MIC range, 16 to >64 ,ug/ml). Similarly, five isolates ofC. albicans and one isolate of C. parapsilosis appearing suscep-tible to flucytosine by the microdilution tests at 24 h (MICrange, 0.125 to 2 ,ug/ml) were judged to be relatively moreresistant to this drug by the reference macrodilution and by the48-h reading with the microdilution tests (MICs, >64 ,ug/ml).Interestingly, we found that for the isolates of C. neoformansvar. neoformans, the agreement between the macro- andmicrodilution methods was higher with the first reading (48 h)of the microplates when testing fluconazole (94 versus 92%).These findings suggest that when testing fluconazole against C.neoformans var. neofornans, the microdilution technique pro-

TABLE 6. General agreement between macro- and microdilutionmethods for four drugs tested against 273 clinical yeasts isolates

vides the advantage of an earlier MIC determination (48 hinstead of 72 h). Similarly, we found that when testing flucon-azole against T. beigelii, the best agreement between themethods is at the first reading of the microplates. Itraconazolehas not been tested previously in comparisons of macro- andmicrodilution results. A good agreement (85.7%) was alsofound with this triazole, with the lowest value for C. parapsilosis(72%) and the highest for C. lusitaniae and C. krusei (100%).The greatest difficult in interpreting susceptibility results for

antifungal drugs, especially the azoles, is the partial growthinhibition, or trailing effect, produced by these drugs. The 1:5dilution (80% inhibition) in the macrodilution tests provides aguide for determining endpoints when the trailing occurs. Ourstudy showed that the use of slightly hazy turbidity to defineazole and flucytosine MICs in the microwells provides goodcorrelation with the results obtained by the reference macrodi-lution tests. Before reading the microplates, it is important toproduce a homogeneous suspension in the wells by agitatingthe microplates for few minutes. This simple procedure im-proves the visualization of different rates of growth in thesesmall volumes of medium. Recently, Rodriguez-Tudela andMartinez-Suarez, using a microdilution technique for testingfluconazole against 92 clinical isolates of C. albicans, reportedthat RPMI 1640 supplemented with 18% glucose facilitates thedefinition of the fluconazole MIC endpoint (35). The sameinvestigators, comparing spectrophotometric (minimum drugconcentration which inhibits 50% of growth) and visual (80%inhibition) reading of the microplates, also found a goodcorrelation between the two different reading approaches (35).Our data indicate that the microdilution test also gives accu-rate MIC determinations by a simple visual reading. Thissuggests that the microdilution method may have clinical utilityin laboratories involved in antifungal susceptibility testing ofyeasts. It must be highlighted, however, that the clinical role ofthese in vitro tests is not well documented, and more studiesare needed to evaluate the in vitro and in vivo correlations ofdrug efficacy.

In the last few years, the development of antifungal resis-tance with prolonged treatment has been reported frequently,mainly in patients with AIDS suffering from recurrent episodesof oral candidiasis (3, 5, 12, 19, 32, 39). There have beenseveral efforts to correlate the in vitro data with the clinicaloutcome in these patients (7, 20, 29, 32, 36, 39). All 100 strainsof C. albicans that we tested in this study were also isolatedfrom the oral cavities of AIDS patients, and this explains thehigh percentage of fluconazole MICs of .64 ,ug/ml. The goodagreement found between the reference macrodilution andmicrodilution techniques in testing fluconazole against C.albicans suggests that both methods are capable of discrimi-

% Agreement between macro- and microdilution methods for:Reading

Fluconazole Itraconazole Flucytosine Amphotericin B

1st 67 52.7 77.7 85.32nd 92 85.7 98.3 96.4

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nating isolates that are relatively more resistant to fluconazolefrom susceptible ones.

In conclusion, this study documents an excellent agreementbetween the macro- and microdilution techniques when testingfluconazole, flucytosine, and amphotericin B. Moreover, ourdata indicate that, by following the recommendations providedby the NCCLS for the broth macrodilution test, both methodscan be used for testing itraconazole as well. Additional multi-center studies are necessary to determine the interlaboratoryreproducibility of this microdilution method.

ACKNOWLEDGMENT

F. Barchiesi is a recipient of a scholarship from Istituto Superiore diSanita, Rome, Italy.

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Comparative ofBroth Macrodilution and Microdilution Techniques … · Malattie Infettive e Medicina...

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