Evaluation of the MICUR system for quantitative antimicrobial susceptibility testing: a multiphasic comparison with reference methods

Vol. 16, No. 1 JOURNALOFCLINICAL MICROBIOLOGY,July1982,p.153-163

0095-1137/82/070153-11$02.00/0

Evaluation of

the MICUR System for Quantitative

Antimicrobial Susceptibility Testing: a Multiphasic

Comparison

with Reference Methods

RONALD N.

JONES,'*

ARTHURL. BARRY,2JUDY

BIGELOW,'

THOMASL. GAVAN,3AND CLYDE

THORNSBERRY4

Departmentof Pathology, Kaiser Foundation Laboratories (Oregon Region), Clackamas, Oregon 970151; Clinical Microbiology Laboratory, University of California, Davis, Medical Center, Sacramento, California

958172;DepartmentofMicrobiology, The Cleveland Clinic Foundation, Cleveland, Ohio441063;and

Antimicrobics Investigations Section, Centers for Disease Control, Atlanta, Georgia303334

Received 29 December 1981/Accepted2 April 1982

Four

laboratories participated

in a three-phase study to evaluate the MICUR

antimicrobial broth microdilution

system (Boehringer Mannheim Diagnostics,

Inc., Houston,

Tex.). The

dried-antimicrobial

agent MICUR system was

com-pared with

a

reference broth microdilution

method (National Committee for

Clinical

Laboratory

Standards) by using

304 recently

isolated

clinical strains and two

collections of

stock or challenge organisms. Of 7,092 minimum inhibitory

concentration (MIC) datum pairs derived from

the

clinical

isolates, 96.6% were

within

an

acceptable

(±1

log2 dilution)

range. MICUR MICs agreed with the

reference broth microdilution

method

MICs in 95.3%

of 6,840 MIC pair

determi-nations performed

on

stock

or

challenge cultures.

The MICUR intralaboratory

reproducibility

within

±1

log2

dilution step for the clinical isolates was 98.4%. The

MICUR

intralaboratory and interlaboratory

reproducibilities for 26 stock cultures were

98.4 and

95.1%, respectively.

For 180 challenge cultures (4,199 MIC pairs)

which

were

included in

the

MICUR

testing to provide a wide variety of

antimicrobial susceptibility and resistance

patterns,the results for

92.5%

were in

close

agreement

with

the

reference broth

microdilution

results. No specific

resistance mechanism

went

unrecognized by this

new

commercial

system. The

MICUR

system

gives comparable

MIC results when evaluated against

the

reference broth microdilution method, and it would be acceptable for

use in

clinical

microbiology

laboratories.

The

availability

of

clinically acceptable

com-mercially prepared broth microdilution

products

for

quantitative

antimicrobial

susceptibility

test-ing enables

most

clinical laboratories

to

easily

use

the

methods

as an

alternative

toagar

dilution

or

tube

dilution

procedures. Systems

containing

dried

antimicrobial

agents

(Sensititre,

3M-MPS,

and

Sceptor)

have

been evaluated and found

to

be

accurate,

reproducible,

and

readily

applica-ble in

clinical

laboratories (5, 7, 8, 10;

E.

H.

Gerlach, manuscript

in

preparation).

Similar

re-sults have been

reported

for one microdilution

system

containing

antimicrobial

agents

frozen

in

broth

(3). Notable

advantages of

dried-antimi-crobial

agent systems over

their

frozen-antimi-crobial

agent counterparts are

longer

shelf

life,

ability

to

utilize different

media,

and

ability

to test

drug combinations.

Also

noteworthy

is the

greater

degree of method standardization

that

can be

accomplished by

a

single

commercial

laboratory

preparing large

batches

of

trays

for

clinical

use than

by

individual clinical

labora-tories each

preparing their

owntrays

(3, 5, 7,

8).

In

this study

we

evaluate the

MICUR system

(Boehringer Mannheim Diagnostics,

Inc., Hous-ton,

Tex.),

anew

commercial dried-drug

micro-dilution product. Three

tray

designs

or panels

(gram

negative,

gram

positive, and urinary)

are

intended for clinical

useto

determine the

mini-mum

inhibitory

concentration

(MIC) of specific

antimicrobial

agents. Four

laboratories

partici-pated in this study (three associated with

medi-cal

centers)

by directly comparing

MICs

ob-tained with the MICUR systempanels and MICs

obtained with reference

trays

prepared

at each

facility.

MATERIALS AND METHODS

Testorganisms. Threephases oftestingcomprised thisstudy.Inphase 1,26stock bacterialstrains were

testedbythetwo susceptibilitytestingmethodson3 separate days at each of three

participating

labora-tories(The Cleveland Clinic Foundation, the Kaiser FoundationLaboratories,and the

University

of

Cali-fornia, Davis,MedicalCenter).These

organisms

(see

153

on February 7, 2020 by guest

http://jcm.asm.org/

154 JONES ET AL.

Tables 2 and3)were selectedtoprovideamaximum

numberof individual MICs within the7 log2dilution range for each antimicrobial agent provided in the MICURsystem panels (3, 5, 8). Onlythose MICsat

leastonewellremoved from thehighorlowend of the concentration range were designated as "on scale".

The reference microdilution (RMD) trays were

pre-pared ateach participating laboratory such that the antimicrobial agent dilutions and MIC ranges were

equivalent to those found in the MICUR system panels. Further details about these bacteria and

ap-plied statistics can be found in previously reported studies(3, 5, 8).

Inphase2,180 stock bacterial strainsweretestedat

the Centers forDisease Control. These strains were

selected from stock culturestoprovideawidevariety ofantimicrobial susceptibilityandresistancepatterns,

e.g., methicillin-resistant Staphylococcus aureus,

1-lactamase-producing Enterobacteriaceae, aminogly-coside-resistant Pseudomonas spp., etc. (8). Each isolate was tested in aurine tray, andthe tray was

determined bytheGramstain result forthe organism. The organisms tested in this challenge phase were

distributedasfollows:98Enterobacteriaceae (16

spe-cies),23 nonentericgram-negative bacilli(6species), 39 Staphylococcus spp., and20 bile-esculin-positive streptococci.

In phase 3, 304 fresh (within 48 h of isolation) clinical bacterial isolates were tested. Isolates were

identifiedtothespecieslevel bythe API systemor a comparable method (2, 4). Each isolate was then testedon2 consecutivedays bythetwosusceptibility testing methods. MICs obtained with the MICUR system were directly comparedwith MICs obtained with the RMD method (1, 9). The Cleveland Clinic Foundation testedgram-negativebacilli(102isolates), the Kaiser Foundation Laboratories tested urinary

tract pathogens (104isolates), and the University of California, Davis,MedicalCentertestedgram-positive strains (98 isolates). In the gram-negative and urine series, no more than 25% of the strains could be Escherichiacoli. Othergeneraandspecies includedby protocol design were Klebsiella spp., Enterobacter spp., Serratiamarcescens, Proteusmirabilis, indole-positive Proteusspp., andPseudomonas spp. Inthe

gram-positiveseries,nomorethan50%of theisolates could be S. aureus. S. epidermidis and enterococci represented themajority ofthe other cocci.

Referencesusceptibilitytests.Thebroth RMDtrays

werepreparedineachofthe four participating

labora-toriesbyusingeither the Dynatech MIC 2000 (Dyna-techLaboratories, Inc.,Alexandria,Va.)ortheQuick SpenseII (Sandy SpringsInstrument Co.,Ijamsville,

Md.).Eachparticipating investigatorreceiveda

com-mon lot of each antimicrobial agent powder with knownpotencyandacommon lotof Mueller-Hinton broth(DifcoLaboratories, Detroit, Mich.). The broth was supplemented with calcium and magnesium to

contain50and 25 mg/liter, respectively(9). The test

inoculawerepreparedinthefollowingmanner.Using

a sterile technique, we inoculated 4 to 5 isolated, morphologically similar bacterial colonies from an

overnight culture (derived from stock culturesor

re-centlyisolatedclinicalstrains) into5mlof Trypticase

soybroth(BBLMicrobiology Systems, Cockeysville, Md.);theinoculated tubeswereincubated for2to6h

at35°C untiltheturbiditywasapproximately

equiva-TABLE 1. Antimicrobial agent concentrations in MICURpanelsafteraddition of 50 ,ul of

cation-supplemented Mueller-Hinton broth

Drugconcna in indicatedpanel

Antimicrobialagent Gram Gram

negative positive Urine Amikacin 0.5-32 0.5-32 0.5-32

Ampicillin 1-64 1-64 2-128

Cephalothin 2-128 1-64 2-128

Gentamicin 0.25-16 0.25-16 0.25-16

Tetracycline 0.5-32 0.25-16 0.25-16

Chloramphenicol 1-64 1-64 NAb

Kanamycin 1-64 1-64 NA

Carbenicillin 8-512 NA 8-512

Cefamandole 2-128 NA 2-128

Cefoxitin 2-128 NA 2-128

Tobramycin 0.25-16 NA 0.25-16

Colistin 0.25-16 NA NA

Clindamycin NA 0.25-16 NA

Erythromycin NA 0.25-16 NA

Methicillin NA 0.25-16 NA

Penicillin NA 0.12-8 NA

Vancomycin NA 0.5-32 NA

Nitrofurantoin NA NA 4-256

Sulfamethoxazole/ NA NA 10-640c trimethoprim

Trimethoprim NA NA 0.5-32

a Base 2logarithmdilution range in

micrograms

per milliliter.

b NA, Notavailable.

cDenotes sulfamethoxazole concentration only, fromaratioof 1 parttrimethoprim to 19 parts sulfa-methoxazole.

lent toor diluted to a 0.5 McFarland turbidity stan-dard. Three milliliters of eachadjusted broth culture wasadded to a 30-ml waterblank and mixed thorough-ly. This dilution was then poured into a disposable inoculum tray and inoculated withaDynatech semiau-tomated inoculator (delivering ca. 1 ,ul to each well containing 100 ,ul of antimicrobial agent broth). The final inoculum approximated 1 x 105 to 5 x 105 colony-forming units per ml.

MICURtestsystem.MICURtestpanels were inocu-lated withthe same standardized inoculum used for theRMD trays. Ten microliters of the adjusted cell suspension (ca. 108colony-formingunits per ml) was addedtoa tubecontaining10 ml ofcation-supplement-ed Mueller-Hinton broth, mixed thoroughly, and poured intoasterile petri dish. A multichannel pipet-torwas used to reconstitute and inoculate each well with 50p.l of inoculum broth.

Afterovernight incubation (16 to 18 h) of the tape-sealed trays, the MICs for both methods were read with a mirror reader and recorded as the lowest concentrationofantimicrobial agent which completely inhibitedvisible growth. These MICs were determined independently by two observers, and disagreements were clarified by a third reader, usually the primary study monitorattheinstitution.

RESULTS

Table 1 lists the antimicrobial agentsand the

dilution range ofeach tested in three different

J. CLIN. MICROBIOL.

on February 7, 2020 by guest

http://jcm.asm.org/

MICUR SYSTEM EVALUATION 155

11 ° b *0 -t ^ oX^0B

flt

r kz

Q

00 0

IAIAIAIAIAZs Zs S (

00~ ~ ~ ~

00~~~~~~ ~ ~~~~~~~~~~~~~~~~~'0

-.- IA IA IA AAI IA IA

0 0000 X X oo oo oo oo-1X° °v,o co

V

VV

VSS

~~~~IA

IA IA IA IA IA IA IA IA IA, IA IA IA IA ~~~~~I AnQ

oo0ooo% 00ooo ooo00oo oo0,,%O,0a ON o0000 X

v v

vv

v

v

v

vv v

" ts,, IA IA IA IA A.A I

00 000000 00 000 000t'000om -A, m m 0000 03

-vvvv r

VIA IA IA IA IA IA IA IA IA IA IA IA IA IA IA IA

0000 0 :~-4h- i00 0000

00a'a7.ttc

J0

rio

oo

oo

os

sS

~IA

A0 IA IA IA

IAIA

IA

IAIA

IA IA IA

IAAIACIA

IIA IAA A IA

Cm

a'ae

000000 00 . ..t 0t4. > '0

vv>v~ ~ ~ > >>> > 35

VIA IA

IAIA

IAlIA

IA IA IA IA IA IA

IA IA

_S&O0

0001a.00 0. 0 0t" o- OOP .0 sOOOa...0

~ji1LAj~j il A

0

o

> QItQCJlCAtALCJ CA AC1Cs LAQ

IA IA IA IA IA IA IA

CDle

ra3

v Vv 3

UJ~~~~~~~~~

V V VV VV VVV m 0

v v v v Q >n

IA V V VV V V V V IA IA IA IA IA IA IA

CD ON aA ONal a' C7% C7,C- DC 0 X

o0o0 0hO%io r00 .0 000000000000o

i LI~LAIAIAIAIIALAAII A AIA

LA LA ~~~~~~~~~~~~~~LAI-' (.A~ LACb.A L-A ~

v

vv

v

v v

z-"t-i --- zz

8N 8s 8 ON3 7 O" oo0oo o o o7oooooooN ooN ^

ZVZ

V

VV IA IA IA IA IA IA IA IA IA IA IA IA IA Z Z

0000 0-0000000000000000

C.JjJj ZZ IAlIA IAIAAIAIAIAIAIAIA IA IAIAZZ L.ALZ Z i,A^LA L ALA A L-AiALA

VOL. 16, 1982

OPr

_.I .

>

on February 7, 2020 by guest

http://jcm.asm.org/

156 JONES ET AL. J. CLIN. MICROBIOL.

F-H H-F--ooo

zz

zzzzzzzzzz

VI VI VIVI VI

V) WVI) VWI VI)

66 0 0666 6m

VI VI VI VI A VI VIVIVIVIVI

AA ~ A

VI VI VlVI r-IrV I VI VIV

A A

VI VI VI VI

A A F-zz ciCi VI VI n . VI VI zz VI VI _r un " W) 6 VI

WI WI U) U)

'.O'01 o 60 ri4

V-0 _-4 IroT-^ en

A A A

zz zz zzzz zz

W) W) t;r; tz;W _E oos

un Un u u U#)n

6666666666D00

VI VI VIVI VIVI VIVI VI VI

VI VI VIVI VI VIVI

VI VI 4 4 VI VI VI VI VI VI

EH F- F-HF-FF F F'

ZZZZZZZZzz e

Vl---q

VlV--l --l '.0Vl

VI VI VI VI VI VI VI VI VI VI -"

F-F-4 1

-zz ~r ~~

F-H 0000 0000

1O

z

z000

-4

E r

ZZ A A A A A A Z Z - "' " e"

00 00

r-11.

r-( "~

A A

en

A A A A

IT IT IT

'IO '.0%D o o '.0

A A A

00

'.0 A.

A A A

c^e^en^^

A A A A A A A A

A A A

ci 'IC C4 ci ci ci

so " £c £- 1Xo "" " enr- r" r^ en en

Ci

A A

"S

,soCi1"

F-- E HF FFFFFFF oNN ot£ x

zz ZZZZZZZZZZ-e m

V- r- --l ---r "C -4 -I ---Ci

VI VI VIVI VI VI VI VI VI VI VI VI VI VI VI VI VI VI VI VI VI

04 W: 04 4 04 a: 0: P4 04 04

EE E u : u UE u u u

~ ~ ~ Z

PN Cq tq Cq cq Cq Cv Cq Cq c

0 H

z

C C.)

z

z

U H w H Li)

z

Li) H Li) H C H H

z

LI)

z

Li) U Li) U U 0 0 E .4 0 co 3 0-4)4) 01

o

-O

0~ 4) _ 4) Cxs 1.. 2 0 *0e ._a. Cd tot (U .Q.2 0_ CL

4).-14U)4_0

4> CZ 40 - O-C) U7 ) 4); X41) 4) _14 cn) H

on February 7, 2020 by guest

http://jcm.asm.org/

MICUR SYSTEM EVALUATION 157

TABLE 4. Comparisonof MICUR and RMD MICs, expressed as MIC ratios, tested at each laboratory and indexed by Gram staincharacteristicsa

Organism group and No. of strains with MICURMIC/RMD MIC ratios of: %at ratios of

laboratoryb

sO.25 0.5 1 2

-4

0.5, 1, and 2

Grampositive

Cleveland 42 141 364 24 5 91.8

Kaiser 44 257 746 32 1 95.8

U.C., Davis 77 250 738 14 1 92.8

Total 163 648 1,848 70 7 93.8

Gramnegative

Cleveland 31 305 668 61 15 95.7

Kaiser 48 427 967 61 9 96.2

U.C.,Davis 48 450 950 61 3 96.6

Total 127 1,182 2,585 183 27 96.2

Allorganisms

Cleveland 73 446 1,032 85 20 94.4

Kaiser 92 684 1,713 93 10 96.1

U.C., Davis 125 700 1,688 75 4 95.0

Total 290 1,830 4,433 253 34 95.3

aAll MICsweretabulated(6,840totaldeterminations)from phase 1 studies.

bCleveland, The Cleveland Clinic Foundation; Kaiser, Kaiser Foundation

UniversityofCalifornia, Davis, Medical Center.

MICUR systempanels. In phase 1, each strain

wastested in MICUR and RMD trays onthree

separate occasions (usually on consecutive

working days) by usingone or more of thetest

panels. Tables 2 and 3 list the modal MICs

obtained from the MICUR and RMD trays.

Directcomparisons weremadeby matching the

MICUR MIC toanRMD MICoriginating from

thesamesubculture, and resultswereexpressed

asMIC ratios(MICUR MIC/RMD MIC). For 26

organisms tested in the first phase (367 modal

MIC pairs), only17MICs(4.6%) deviated from

theRMD MIC bymore thanonedilution

inter-val. Streptococci accounted for 11 of the

dis-crepancies, all with drugs of no clinical

rele-vance, e.g.,aminoglycosides, carbenicillin, and clindamycin.

Table4shows theratios between paired

MI-CUR and RMDMICs. Allon-andoff-scale MIC

ratiosprovided 6,840 comparisons; 4.7%of the

MIC datum pairs had MIC ratios beyond the

acceptable ±1 log2 dilution range. The use of

only on-scaleratios for theanalysisresultedina

93.8% agreement between methods. The

intra-laboratory variability was analyzed for each

method by comparing the MIC results of each

trialexpressedin base 2logarithmdilutionsteps

(Table 5).Theintralaboratory

reproducibility

for

MICURrangedfrom99.2%atthe Kaiser

Foun-dation Laboratories to a low of 97.0% at The

Laboratories; U.C., Davis,

TABLE 5. Summary of phase 1 intralaboratory variations for the MICUR and RMD procedures tested at The Cleveland Clinic Foundation, Kaiser

Foundation Laboratories, and the University of

California,Davis, Medical Center'

% MICs atlog2dilution %with MIC method and variationof: acceptable

laboratoryb 2 1 variations(s1

laborator-'~3 2 1 0 log2 dilution) MICUR

Cleveland 0.5 2.4 21.0 76.0 97.0 Kaiser 0.1 0.7 15.7 83.5 99.2 U.C., Davis 0.1 1.4 14.3 84.2 98.5 Total 0.2 1.4 16.5 81.9 98.4 RMD

Cleveland 0.2 0.9 16.8 82.0 98.8 Kaiser <0.1 0.8 9.3 89.8 99.1 U.C, Davis <0.1 0.8 14.4 84.7 99.1 Total 0.1 0.8 13.1 86.0 99.1

a Totals represent all data, including on- and off-scale MIC results. Theuseofonlytheon-scaleMICs didnotresult inanyappreciable changefromthe cited analyses (97.8 to 98.9o of values at ±1 or 0 varia-tions). The MICs of the three trials were directly

compared,andthe variationswerethenexpressed in

lg2

dilutions.

Equal

MIC results between trials were

assigneda0variation value.

b See Table 4, footnoteb, for laboratory

names.

VOL. 16,1982

on February 7, 2020 by guest

http://jcm.asm.org/

158 JONES ET AL.

TABLE 6. Summaryof phase 1interlaboratory variationsfor the MICUR and RMD procedures tested at TheClevelandClinic Foundation,Kaiser

Foundation Laboratories and theUniversity of

California, Davis,MedicalCenter'

% MICsatlog2dilution % with

MIC method and variation of: acceptable

laboratoryb 2 1 variations(s1

laboratory"23 2 1 0 log2dilution) MICUR

Cleveland 0.8 5.8 33.6 59.8 93.4 Kaiser 0.3 4.5 32.6 62.5 95.1 U.C., Davis 0.6 2.9 31.4 65.0 96.5 Total 0.5 4.5 32.5 62.5 95.0

RMD

Cleveland 0.3 1.3 20.1 78.3 98.4 Kaiser 0.3 1.0 12.6 86.1 98.7 U.C., Davis 1.0 1.4 19.0 78.6 97.6 Total 0.5 1.2 17.2 81.0 98.2

aTotals represent all data, including on- and off-scale MICs. Thepresentationofonlytheon-scale MIC

pairswouldnotresult inasignificantdifference from

the datapresented. Theinterlaboratory analyses com-pared the results of allof the trials ateachfacilityto

theall-laboratorymode(ormedian)shown in Tables 2

and 3.Total agreementwasgivena0variation value.

bSee Table 4, footnoteb,forlaboratorynames.

Cleveland

Clinic Foundation. The RMD method

had

agreater

frequency

of absolute

agreements

among

triplicate

tests:

86.0%

versus

81.9%

for

MICUR.

Although this

was a

small

difference,

it

was

significant

(P<

0.05).

Examination

of

varia-tions

at

each

laboratory showed that the

most

variable results

were

obtained

at

The

Cleveland

Clinic Foundation with amikacin when MICUR

was used

(93.5%),

at the

Kaiser

Foundation

Laboratories with colistin when

the RMD

meth-od

was

used

(94.4%),

and

at

the

University of

California,

Davis, Medical Center with colistin

when

MICUR was used (94.4%). Colistin had

the least reproducible MIC by both methods for

all participants; 5.3% of the on-scale MICswere

outside of the acceptable range.

Table 6 summarizes the

interlaboratory

com-parisons. The dataweretabulatedbycomparing

theMIC results from each laboratory with the

overallMIC mode for that

organism-antimicro-bialagentcombination (Tables 2 and 3).

Varia-tionswereexpressed in base 2 logarithm

dilution

step deviations from the mode, i.e.,

0,

1, 2, or

.3. The overallinterlaboratory reproducibilities

forthe MICUR and RMD methods were

excel-lent: 95.0 and98.2%, respectively (P<0.05). Of

the nine drugs with >10% interlaboratory

vari-ability, eight occurred in the MICUR system.

Thelowest reproducibilitieswereforgentamicin

(86.2%), carbenicillin (88.2%), and colistin

(88.9%), allwhen MICURwasused. The

repro-ducibility for the RMD method was .95.8%for

all

drugs.

In phase 2 of the evaluation, 180 challenge

bacteria from the stock culture collection of C.

Thornsberryatthe Centers for DiseaseControl,

Atlanta, Ga., were tested. Each organism was

tested intwotray types;gram-negative isolates

were tested in gram-negative and urine trays,

andgram-positive isolates were tested in

gram-positive and urine trays. Table 7 shows the

percentages of MIC datum pairs for each

orga-nism type that were within the acceptable ±1

log2 dilution range. Consistent with previous

studies, the best comparisonswerefoundamong

thestreptococci (95.0% with ratios of 0.5, 1, or

2). Staphylococci showed the poorest

correla-tion(90.6%), principally duetoavariation of >1

log2dilution with

amikacin, ampicillin,

and

tet-racycline. With fewexceptions,most

organism-antimicrobialagent combinations demonstrated

a trend toward lower MICs with the MICUR

system. Because colistin consistently yielded

poor correlation with both methods, phase 2

datawereretabulated after excludingthe colistin

data. As an example, 50 of 121 colistin MIC

comparisons were .2 log2 dilutions apart. Of

TABLE 7. Comparisons of MICUR MIC/RMD MIC ratios from phase 2a

Organismgroup (no. No. of strainswith MICUR MIC/RMD MIC ratioof: %with acceptable

tested)

-0.25

0.5 1 2 -4 ratiosb(0.5, 1,or2)

Staphylococci(39) 58 235 567 46 30 90.6

Streptococci (20) 20 122 302 32 4 95.0

Enterobacteriaceae (98) 130 587 1,382 124 31 92.9

Pseudomonasl

Acinetobacter (23) 31 114 351 23 10 92.2

Total(180) 239 1,058 2,602 225 75 92.5

aPhase 2involved the challenge organisms processed at the Centers for Disease Control (by C.

Thornsberry)

andpossessing known resistance to antimicrobial agents. Colistin data were excluded (see text). Matched MIC pairswereobtained by using both on- and off-scale MIC results.

bInitialscreening of phase 2 organisms only. Repetitive testing, especially for colistin variations, markedly

reduced the significant variations.

J. CLIN. MICROBIOL.

on February 7, 2020 by guest

http://jcm.asm.org/

MICUR SYSTEM EVALUATION 159

TABLE 8. Organisms in phase 2(Centers for Disease Control)demonstratingconsistent MIC discrepancies

betweenthe MICUR and RMD systems

MIC

(>jg/ml)

Resistance Organism (Accession no.) Antimicrobial agent MIC system Initial Repeattrial

Repeat

tnal mechanismoftest

trial A B strain

Staphylococcusaureus Methicillin MICUR 8 >16 8 Heteroresistant

(1072)a RMD 2 2 2

Cephalothin MICUR 8 8 4

RMD '1 '1 '1

S. aureus(1120) Ampicillin MICUR 32 64 64 Penicillinase

RMD 128 >128 >128

S.epidermidis(77-35630) Carbenicillin MICUR 16 16 8 Pencillinase

RMD 64 64 32

Serratia marcescens Trimethoprim MICUR 2 2 2 ,B-Lactamase

(1096)b

RMD 8 8 8

Enterobacter aerogenes Trimethoprim/ MICUR '10 20 20 1-Lactamase

(1001) sulfamethoxazole RMD 40 160 320

E.cloacae(1088) Amikacin MICUR 8 16 16

P-Lactamase

RMD 1 2 2

Escherichia coli (1008) Trimethoprim MICUR 32 >32 >32 Unknown

RMD <0.5 8 4

Klebsiella pneumoniae Cephalothin MICUR 2 4 4 ,B-Lactamase

(1171) RMD 8 16 16

Pseudomonas maltophiliaCarbenicillin MICUR 128 128 128

3-Lactamase

and

(107647) RMD 512 >512 >512 permeability

barrier

aOne additional S. aureus strain (1052) with intermediate MICs by MICUR and susceptible MICs by the RMD

method for cephalothin only wasfound.

bOne additional challenge strain (S. marcescens 1109) had fourfold lower MICs of trimethoprim by the MICUR method.

c Data areexpressedastheMIC of sulfamethoxazole only.

those

50

discrepant

strains, 30

were

randomly

selected for

repetitive

testing (two trials); 20 of

the 30

remained

discrepant,

most

with lower

MICUR MICs. In

contrast,

60

non-colistin

vari-able

MICs

were

retested,

with

20%o

remaining

outside the

acceptable

±1

log2

dilution

range.

Thus, the

true

variability

between

systems

would be only

1.5%,

with the other results being

only

statistical

errors

resulting

from the

poor

reproducibility of colistin

MICs with both

sus-ceptibility testing

methods.

The variable

MICs results that

were

consist-ent

in

repetitive testing

are

shown

in

Table

8. Five

of the

pairs

were

for

P-lactam

drugs tested

against

methicillin-resistant,

penicillinase-pro-ducing

staphylococci.

The 17

methicillin-resist-antS. aureus

isolates

were

easily

recognized

by

the

MICUR

system,

but

with the RMD

method,

1

resistant strain

appeared

to

be

susceptible.

The

remaining

discrepancies

were

randomly

spread

amongnumerous

species

and

antimicro-bial

agents.

Interpretive

errors

between the

MI-CUR

system

and the RMD method caused

by

these

variations

were rare,

i.e.,

very

major

er-rors

(false

susceptible)

=

0.12%,

major

errors

(false

resistant)

=

0.26%,

and

minor

errors =

0.38%.

Several

organism-antimicrobial

agent

combinations

were more

often associated with

such

interpretive

discrepancies.

These

combina-tions

were:

Enterobacteriaceae and

ampicillin,

cephalothin,

nitrofurantoin,

or

trimethoprim;

nonenteric bacilli

and

carbenicillin;

staphylo-cocci and

amikacin,

ampicillin, cephalothin,

or

tetracycline;

and

streptococci

and

cefamandole. In

phase 3,

304

recently isolated clinical

strains

were tested at three

medical

centers.

Table

9

shows

the

results

tabulated

as

MICUR

MIC/RMD MIC ratios for all

organism

groups

tested

against

the 20

antimicrobial

agentsatthe

three

participating hospitals.

The percentage

(96.1%)

of

acceptable

ratios

(0.5, 1,

and

2)

found

for

the

clinical isolates

was

slightly superior

to VOL.16,1982

on February 7, 2020 by guest

http://jcm.asm.org/

TABLE 9. Comparison of phase 3 clinical isolates by using 7,092 MICUR MIC/RMD MIC ratiosa

Laboratoryb

(no.of No. (%) of strains with MICUR MIC/RMD MIC ratio of:

Organismgroup comparisons) -0.25 0.5 1 2 -4

Staphylococcic Kaiser(192) 3 27 159 3 0

U.C.,

Davis

(1,824)

64 498 1,221 32 9

Total 67(3.3) 525(26.0) 1,380(68.6) 35(1.7) 9(0.4)

Streptococcid Kaiser(336) 12 107 215 2 0

U.C., Davis(582) 27 192 299 10 0

Total 39(4.5) 299(34.6) 514(59.5) 12(1.4) 0(0.0)

Enterobacteriaceaee Cleveland(1,958) 58 573 1,193 119 15

Kaiser(1,752) 42 453 1,219 37 1

Total 100(2.7) 1,026(27.7) 2,412(65.0) 156(4.2) 16(0.4)

PseudomonaslAcinetobacter Cleveland(286) 0 46 222 17 1

groupf

Kaiser(216) 9 50 154 3 0

Total 9(1.8) 96(19.1) 376(74.9) 20(4.0) 1 (0.2) Allorganisms(7,092) 215(3.0) 1,946(27.4) 4,682 (66.0) 223(3.1) 26(0.4)

aAll ratioswereMICURMIC/RMD

MIC,

totalling 7,092

MIC datum

pairs.

Theuseofon-scale

MICs,

i.e.,

MICs .1 log2 dilution removed from the extremes of thelog2 dilution sequence (2,882 MIC pairs), did not significantly alter theanalysis(96.3%acceptable).

bSeeTable 4, footnoteb,forlaboratorynames.

cIncludes the following species (with the numbers obtained at The Cleveland Clinic

Foundation,

Kaiser Foundation Laboratories, and University of California, Davis, Medical Center, respectively, given within parentheses): S.aureus(0,2,49)and S.epidermidis orS.saprophyticus (0, 6, 27).

dIncludes S.faecalis

(0,

13,22)and otherenterococci(0,2, 0). (Seefootnotec.)

eIncludesE.coli(25,29, 0),K.pneumoniaeandK.oxytoca(15, 22, 0),E.aerogenes andE.cloacae(14, 3, 0), P.mirabilis (10, 10, 0),P.rettgeri(2,1,0),Morganella morganii (6, 1, 0),Citrobacter diversus(0, 2, 0),Serratia spp.(15, 2, 0),P. vulgaris(2, 1, 0), P.stuartii(0,1, 0),andSalmonella spp.(0, 1, 0). (Seefootnotec.)

fIncludesP.aeruginosa (10,9, 0),P.fluorescens(1, 0, 0),andA. calcoaceticussubsp.anitratus(2, 0, 0). that

found for the stock

organisms

tested in

phase

1

(95.3%)

and

the

challenge

organisms

tested in

phase

2

(92.5%).

Testing of organism

groups

against

19

antimicrobial

agents

(exclud-ing

colistin) showed (Table 9)

a

correlation of

96.9%

forEnterobacteriaceae,

98.0%

for

nonen-teric

gram-negative bacilli,

96.3%

for

Staphylo-coccus spp.,

95.5%

for

streptococci,

and96.6%

for all of the

organisms

tested. MIC ratios for

each

antimicrobial

agent (Table 10) showed the best

correlation

between methodsfor

trimetho-prim/sulfamethoxazole

(100%),

tetracycline

(99.2%), clindamycin (99.0%),

and cephalothin

(99.0%).

The poorest

correlation

was observed

for colistin

(78.9%).

The clinical

isolates

demonstrated the same

trend toward

having

lower MICs with the

MI-CUR system than with the RMD method, as was

noted

for

the stock strains in phases 1 and 2.

This skewing

of

results between methods was

most

clearly

seen

(Table 10) with amikacin,

chloramphenicol, gentamicin,

methicillin,

nitro-furantoin, tobramycin, and vancomycin, for

which

the results

represented

a modal

shift

of

.0.4

of

a

log2

dilution interval between

meth-ods. Of 114

clinical

on-scale

MIC

datum

pairs

differing

by greater than

±1

log2

dilution

step

(ratios of

c0.25 or

.4),

99 had

significantly

lower MICs with the MICUR system than with

the RMD method and 15 had

higher

MICs

with

the MICURsystem than with the RMD method.

Thirteen

(0.4%

of on-scale MIC datum pairs)

very majorerrors were

discovered,

i.e., a

sus-ceptible

MICUR MIC

and

a resistant RMD

MIC.

The

combinations

of organism and drug

that most

often

produced significant error were

as

follows:

Staphylococcus

spp. and

aminogly-cosides, four; Streptococcus

faecalis and

genta-micin,

three; and S. faecalis and

cephalothin,

two.

Major interpretive

errors (false resistant by J. CLIN. MICROBIOL. 160 JONES ET AL.

on February 7, 2020 by guest

http://jcm.asm.org/

MICUR SYSTEM EVALUATION 161

TABLE 10. Resultsofallclinicalorganisms tested against 20 antimicrobial agents at the three collaborating laboratoriesa

Antibiotic _testNo._pairsof _-0.25No.of strains with MICUR_0.5 MIC/RMD₁ MIC ratio of:₂

₂₄

Ampicillin 608 23 167 404 11 3

Carbenicillin 412 3 66 312 29 2

Cephalothin 608 6 102 483 17 0

Cefoxitin 412 3 57 338 11 3

Cefamandole 412 2 27 357 22 4

Kanamycin 400 17 97 267 17 2

Amikacin 608 41 230 315 20 2

Gentamicin 608 51 258 291 6 2

Tobramycin 412 25 211 168 7 1

Chloramphenicol 400 10 215 170 5 0

Tetracycline 608 3 121 436 46 2

Penicillin 196 4 31 147 13 1

Methicillin 196 10 75 109 1 1

Erythromycin 1% 2 17 171 4 2

Clindamycin 1% 1 21 173 0 1

Vancomycin 196 5 145 46 0 0

Nitrofurantoin 208 5 82 114 7 0

Trimethoprim/sulfamethoxazole 208 0 5 201 2 0

Trimethoprim 208 4 19 180 5 0

Subtotal 7,092 215 1,946 4,682 223 26

Colistin 204 38 67 84 10 5

Total 7,2% 253 2,013 4,766 233 31

aRatios weretabulated as MICUR MIC/RMD MIC for 304 organisms. All data pairs, including on- and off-scaleMICs,wereused. Numbers inboldface type represent a modal skewing of.0.4of alog2dilution interval between methods.

the MICUR system) were noted with only six

isolates (0.2%), one-half of these being

Entero-bacteriaceae and ,B-lactam combinations.

Finally, Table 11 shows the intralaboratory

reproducibility data for 3,675 MICUR MICs and

3,656 RMD MICs.Thevariationofthe first MIC

whencomparedtothesecondMIC(determined

within 48 h) is expressed in base 2 logarithm

dilution intervals. MICUR MICsforenterococci

were 100% reproducible (+1 dilution from the

initial MICUR MIC). RMD MICs were less

reproducible than MICUR MICs only for the

enterococci (99.4%)but showed reduced

varia-tionfor

staphylococci

(99.6%),

Enterobacteria-ceae(99.0%), and nonentericgram-negative

ba-cfili

(99.3%) and an overall

reproducibility

of 99.3%.

DISCUSSION

The number of clinical microbiology

labora-tories using quantitative antimicrobial

suscepti-bilitytestingmethods hasmarkedlyincreased in

recentyears. The statistics foreach method as

monitoredbythe College ofAmerican

Patholo-gists Surveys show that 22.4% of the

labora-toriesused dilution methodsinearly 1980,and at

least 64.6%of these usedacommercial

microdi-lution method (6).

The percentage

increased

to

27 to

28% in the first

quarter

of

1981, and again

the

vast

majority

utilized

frozen and dried

com-mercial products

(Survey Critiques

D-06

and

B-11,

1981

College

of American Pathologists

Bac-teriology Surveys). Evaluations of the

commercial

products

have

generally

been

favor-able, and their

widespread

availability

has

signif-icantly contributed

to

this

emerging

trend

to-ward

dilution procedures (3, 5, 7, 8, 10; Gerlach,

manuscript in

preparation).

The

College of

American

Pathologists

Surveys have shown

that

these

commercial

products

have

performed

well

on

their

challenge

samples

and

routinely

demon-strate

reproducibility

equal

or

superior

to

that

obtained with broth

microdilution

trays oragar

plates manufactured

by

individual laboratories.

This

finding

was not

unexpected

since

the

stan-dardization and

reproducibility

of

a

well-con-trolled

commercial

product

certified

by

the Bu-reau

of Medical

Devices,

U.S. Food

and

Drug

Administration,

have

resulted

in

excellent

and

improving

interpretive

accuracy

for

the

disk

diffusion

tests

(6).

With the recent increase in the

number

of

new

antimicrobial

agents

and

the

complexity

involved in

treating

infected

pa-tients,

it has becomemore

important

tohave the VOL.16, 1982

on February 7, 2020 by guest

http://jcm.asm.org/

TABLE 11.

Reproducibility

ofintralaboratory MICUR and RMD MICs for 304recentlyisolated clinical strains testedatthreemedicalcenters'

No. (%)ofrepeatMIC resultsatlog2 %withacceptable Organismgroup(no.

tested)b

MIC method dilution variation of: variation(-1 log2

0 1 .2 dilution)

Staphylococci

(84) MICUR 878(86.4) 127(12.5) 11(1.1) 98.9

RMD 903(89.6) 100(10.0) 5(0.5) 99.6

Streptococci

(36) MICUR 377(85.5) 64(14.5) 0(0.0) 100.0

RMD 386(89.8) 41(9.6) 3(0.7) 99.4

Enterobacteriaceae (162) MICUR 1,503 (76.9) 411 (21.0) 40(2.1) 97.9

RMD

1,615

(82.7)

320

(16.3)

19

(1.0)

99.0

Pseudomonas/Acinetobacter MICUR 207(78.4) 50(18.9) 7(2.7) 97.3

group (22) RMD 231(87.5) 31(11.8) 2(0.8) 99.3

All

organisms

(304) MICUR 2,965(80.7) 652(17.7) 58(1.6) 98.4

RMD 3,135 (85.7) 492 (13.6) 29(0.7) 99.3

aAllon-and off-scaleMIC

comparisons

weretabulated(3,675MICdeterminations). Theuseofonlyon-scale MIC

pairs

didnot

significantly

alter theresults.

bSee Table9,footnotesc, d, e, and

f,

for thelistingofbacterialspecies tested.

wider base of

antimicrobial

susceptibility

infor-mation offered

by

the

dilution

test

methods.

The

MICUR

system

now

joins

Sensititre and

Sceptor

as an

acceptable

dried-antimicrobial

agent

microdilution

product capable

of

produc-ing

MIC results

equivalent

to

those

produced by

the

reference

procedure

(5, 7, 8, 10).

Previous

evaluations also showed that the

frozen-antimi-crobial

agent system

from

Micro-Media

Systems

correlated

excellently

with the

reference broth

microdilution and classic tube dilution methods

(3).

AU

products

can

be

deemed

comparable,

differing only

in

microdilution broth

volume,

method of

inoculation,

quality

control

proce-dures,

and

acceptable

shelf life.

In

this

report,

the

MICUR

system showed

correlations with the

RMD

method

(±

1

log2

dilution

step)

of

95.3, 92.5,

and

96.6% for the

three

phases

of the

study.

These results

were

remarkably

similar

to

those found for

some

other

dried-antimicrobial

agent

products

(5, 7,

8).

In

all

study

phases

a

definite trend

towarda

lower MICUR MICwas detected. A

contribut-ing

factor could

be the common

manufacturing

practice

of

filling

the wells with antimicrobial

agent

at 100 to

130% of

stated

potency.

RMD

trays were

prepared

to contain 100% of the

target concentrations

o-ily.

The

interlaboratory

reproducibility

was assessed in

phase

1 at

95.0%',

avalue

comparable

tothose for

Sceptor

(97.6%),

3M-MPS

(96.2%),

and

the

Micro-Media

Systems

product

(96.0%).

Intralaboratory

vari-ability

was

calculated in

phase

1 and

phase

3.

Again,

the

MICUR

system

had minimal

varia-tions,

with

only

1.6% of MICs outside

the

ac-ceptable

range

in

each

phase. Intralaboratory

reproducibility

data

for other

commercial

prod-ucts were as follows: Micro-Media

Systems

product (stock strains), 96.0%;

Sensititre

(clini-cal

strains), 93.9%;

Sceptor (stock strains),

97.6%;

Sceptor

(clinical strains), 96.9%;

and

3M-MPS

(stock strains),

97.7%

(3, 5, 7, 8;

Ger-lach,

manuscript

in

preparation).

"Skip

patterns"

were

encountered

in this

protocol,

but

ata

frequency

lower than that

seen

in the

Sceptor

or

Sensititre evaluations

(5, 7, 8).

The

rate

of

skips

was

minimized

by

careful

pipetting procedures,

yet

a

high

rate was

still

noted for the

colistin well series. This

finding,

coupled

with the

poor

correlation between

meth-ods and

poor

reproducibility,

suggests

the

omis-sion of this

rarely

used

antimicrobial

agent

from

the

test

panels

(gram

negative only).

A very

difficult

set

of

organisms

was

used in

phase

2 to assess

the

ability

of the

MICUR

system

to

accurately

categorize

strains

as

sus-ceptible

or

resistant.

Most

significant

discrepan-cies between the MICUR and RMD MICs for the

clinical isolates

were for

organism-antimi-crobial

agent combinations of

questionable

clini-cal

relevance. The

very

major

MICUR

system

errors

(false

susceptibility)

were

generally

due

to

low

aminoglycoside

MICs

when

testing

gram-positive

cocci.

We would

consider

the

MICUR

system

equivalent

to

other commercial

and ref-erence

broth

microdilution methods

and

accept-able for

routine

orselecteduse

by

clinical

micro-biology

laboratories.

LITERATURE CITED

1. Barry, A.L. 1976. The antimicrobic susceptibility test:

principlesandpractices. Lea &Febiger, Philadelphia.

2. Barry,A.L.,R. E.Badal,and L.J.Effinger.1981. Identi-fication of Enterobacteriaceae in microtubetest panels.

Lab.Med. 12:546-550.

162 JONES ET AL. J.CLIN. MICROBIOL.

on February 7, 2020 by guest

http://jcm.asm.org/

MICUR SYSTEM EVALUATION 163 3. Barry, A. L., R. N. Jones, and T. L. Gavan. 1978.

Evalua-tion of the Micro-Mediasystemfor quantitative antimicro-bial drug susceptibility testing: a collaborative study. Antimicrob.AgentsChemother. 13:61-69.

4. Fuchs, P. C.1976. Thereplicator method for identification and biotyping ofcommonbacterial isolates. Lab. Med.

6:6-11.

5.Gavan,T.L.,R. N.Jones,andA. L.Barry.1980. Evalua-tionof the Sensititresystemfor quantitative antimicrobial drug susceptibility testing: acollaborative study.

Antimi-crob. Agents Chemother. 17:464-469.

6.Jones,R. N.1981. Status of the art: alookat thepast, presentand antimicrobialsusceptibility testing trends

co-monitored by the CAP Laboratory Proficiency Surveys,

p. 83-90. In H. M. Sommers (ed.), The 1979 Aspen Conference ProceedingsonClinical Relevance in Micro-biology. College of American Pathologists, Skokie, Ill. 7. Jones, R. N., T. L. Gavan, and A. L. Barry. 1980. The

evaluation of the Sensititre microdilution antibiotic

sus-ceptibilitysystemagainstrecentclinical isolates:a three-laboratorycollaborative study. J. Clin. Microbiol. 11:426-429.

8. Jones, R.N., C. Thornsberry, A. L. Barry, and T. L. Gavan. 1981. Evaluation of the Sceptor microdilution antibiotic susceptibility testing system: a collaborative investigation. J. Clin. Microbiol. 13:184-194.

9. National Committee for Clinical Laboratory Standards. 1980. Proposed standard, M7-P. Standard method for dilution antimicrobial susceptibility tests for bacteria whichgrowaerobically. National Committee for Clinical

Laboratory Standards, Villanova, Pa.

10. Phillips, I., C. Warren, and P. M. Waterworth. 1976. Determination ofminimuminhibitory concentrations by the Sensititre system, p. 78. In H. H. Johnson and

S. W. B. Newsum (ed.), Second International Sympo-siuminRapidMethodsand Automation inMicrobiology. Learned Information(Europe) Ltd., Oxford.

VOL. 16, 1982

on February 7, 2020 by guest

http://jcm.asm.org/