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Comparison of a radiometric method (BACTEC) and conventional culture media for recovery of mycobacteria from smear negative specimens

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JOURNALOFCLINICALMICROBIOLOGY, Aug. 1983,p.384-388

0095-1137/83/080384-05$02.00/0

Copyright C 1983, AmericanSocietyforMicrobiology

Vol.18, No. 2

Comparison

of

a

Radiometric

Method

(BACTEC)

and

Conventional Culture

Media

for

Recovery

of

Mycobacteria

from

Smear-Negative Specimens

MARGIE A.MORGAN, CARL D. HORSTMEIER,DONALD R. DEYOUNG, AND GLENN D. ROBERTS*

MayoClinic and Mayo Foundation, Rochester, Minnesota 55905

Received 29 November1982/Accepted 18 April 1983

The BACTEC system and three conventional media (Middlebrook 7H10,

selective Middlebrook 7H11 [S7H11], and Lowenstein-Jensen [LJ]) were

com-pared for their mean recovery times and recovery rates of mycobacteria from

acid-fast, smear-negative clinical specimens. Of the 71 smear-negative,

culture-positive specimens recovered from 2,165 submitted smear-negative cultures, the

BACTEC system detected 71.8%, compared with 88.7% for the conventional

three-mediumsystem.When mediawereindividually compared,BACTEC

medi-um(Middlebrook7H12)wasmoresuccessful in recovering mycobacteria (71.8%)

thanwas LJ (62%), Middlebrook medium 7H10(55.9%), orMiddlebrook S7H11

medium(52.1%). Middlebrook 7H11 mediumcontaining sodium selenatewas also

evaluated and didnotincreasethe recovery rate or decrease the recovery time of

mycobacterial species when comparedwith LJ, Middlebrook 7H10, S7H11, and

7H12media. Themeandetection time for theBACTEC systemwasless than that

by conventional methods for the seven species of mycobacteria recovered.

Detection times for Mycobacterium tuberculosis on the BACTEC system and

conventional cultural systems were13.7 and 26.3 days, respectively.

The BACTEC system (Johnston

Labora-tories, Inc.,Cockeysville, Md.)hasbeen

report-ed to be valuable for the rapid detection of

clinically important mycobacteria. The system

uses Middlebrook7H12 broth medium

contain-ing14C-labeled palmitic acidfortheradiometric

detection ofmycobacterialgrowth and has been

showntobemoresuccessful indetecting

myco-bacteria than conventional methods involving

only one medium (C. D. Horstmeier, D. R.

DeYoung, K. A. Doerr, and G. D. Roberts,

Abstr. Annu. Meet. Am. Soc. Microbiol. 1982.

C187,p. 302).

Previous studies reported the BACTEC

sys-tem tobe assuccessfulasconventionalculturing methodsin detecting growth in specimens

hav-ing apositive direct smearfor acid-fast bacilli.

These studies also revealed that the BACTEC

system detected mycobacterial growth more

rapidly than did conventional cultural methods

(Horstmeier et al., Abstr. Annu. Meet. ASM,

1982,C187;N.Goodman,H.Larsh,T.Lindner, M. McGinnis, K. McClatchy, and G. Roberts, Abstr. Intersci. Conf. Antimicrob. Agents Che-mother. 21st,Chicago, Ill., abstr.no.96,1981).

It has been reported that detection times for

mycobacterial growth in acid-fast

smear-posi-tive specimens, as detected by the BACTEC

system, varied among the five medical centers

that evaluatedthe method, witha meanof8.36

and 5.4 days for Mycobacterium tuberculosis

and mycobacteria other than tubercle bacilli

(MOTT), respectively (Goodman et al., 21st

ICAAC, abstr.no.96).Meandetectiontimes for

M. tuberculosis and MOTT were 13.2and22.8

days, respectively, as determined by

conven-tional methods.

A specimen with a negative acid-fast smear

and apositive cultureformycobacteriaisnot an

unusual occurrence inaclinicallaboratory.The present study evaluated the BACTEC system

and conventional methods for detecting

myco-bacteria in specimens with a negative acid-fast

smearandapositiveculture. Middlebrook7H12

(BACTEC) mediumwascompared with

Lowen-stein-Jensen (LJ), Middlebrook 7H10, and

Middlebrook S7H11 mediafor comparative

re-covery rates and times of seven species of

mycobacteria foundinclinical specimens.

Mid-dlebrook 7H11 containing sodium selenate was

also compared with LJ, Middlebrook 7H12,

7H10, and 7H11 for the recovery times ofM.

384

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MYCOBACTERIA RECOVERY BY BACTEC AND OTHER MEDIA 385

BACTEC | =71.8%

0) 0)

CONVENTIONAL =88.7%

Q

0D

FIG. 1. Comparison of the number of

mycobacte-ria(excludingM. gordonae)recovered from 71 acid-fast, smear-negative specimensbythe BACTEC and conventional methods.

tuberculosis and MOTT. Sodium selenate was

previously reportedto enhance thegrowthof M.

tuberculosis (1).

MATERIALSANDMETHODS

Acid-fast smear-negative specimens, excluding

ce-rebrospinal fluid and urine, submittedto our labora-toryduring a3-month period (June toAugust, 1981)

for routinemycobacterialculturewereincluded inthis

study. Of 2,165 specimens, 71 had negative direct acid-fast smears, butmycobacteriawererecoveredby

either the conventional or the radiometric method.

Specimensfrom whichMycobacteriumgordonaewas

recoveredwere notincluded in this group. Tissue and body fluidsprocessed for mycobacterial culturewere

inoculated directly onto culture media. Respiratory

tract specimens andgastric washingswereprocessed by using2% NaOH decontamination and

centrifuga-tion (3). The BACTEC method used liquid Middle-brook 7H12(Johnston Laboratories, Inc.)containing

"C-labeled palmitic acid. The conventional cultural

technique used three media: Middlebrook 7H10, Middlebrook S7H11 containing polymyxin B (20

,ug/ml), trimethoprimlactate (20,g/ml), and carbeni-cillin (50 ,ug/ml), and two LJ slants (Difco Labora-tories). Selenate 7H11 containing 5 ,g of sodium selenate per ml (Diagnostics, Inc., St. Paul, Minn.) was also evaluated in this study. The Middlebrook 7H10, 7H11, and S7H11 mediawereinoculated with 0.5 mlof concentrated specimen, and twoLJ slants

wereinoculatedwith 0.25 ml each. The 7H12 medium wasinoculated with 0.1 ml of concentrated specimen according to the manufacturer's instructions. A small amountof sediment from the concentratedspecimens was used to prepare smears forauramine-rhodamine staining. The petri dishes containing 7H10, S7H11,

andselenate 7H11 media were sealed inpolyethylene, C02-permeable plastic bags, and allmedia, including LJslants, were incubated for 8 weeksat35C in an

atmosphere of 5 to 10% CO2. Conventional media wereexamined everythirdday for visibleevidence of growth until week 4ofincubation, when they were examinedonceweekly.BACTEC bottleswerereadby using a BACTEC model 460 every third day until week 4ofincubation; then theywerereadonceweeklyfora

totalof 8 weeks. BACTEC bottles withagrowth index reading of -10 were considered to bepositive. Posi-tivebottles were read daily until areading of 100was

reached, and then samples of each medium were used to prepare acid-fast stains and subcultures on LJ medium. Subcultures were incubated at 35°C in an atmosphere of 5 to 10%oC02 until growth was suffi-cientfor biochemical testing.Mycobacteriawere iden-tifiedby conventional methods (2, 3).

RESULTS

A total of 134 mycobacteria were recovered

from2,165 acid-fast, smear-negative specimens

submitted for culture. Sixty-threeisolates of M. gordonae were included; however, these were notconsidered in theanalysisof the data. Bacte-rial contamination appeared in 78 of 2,165

(3.6%) specimens cultured for mycobacteria.

Whencontamination occurred in the 7H12 broth

medium,the broth wassubculturedonto ablood

agar plate and 7H10 medium. Mycobacteria

were recovered from three contaminated

speci-mens from the smear-negative group. Six cul-tures from threepatients yielded morethan one

organism. M. gordonae was the second orga-nism in allexcept two cultures inwhich

Myco-bacteriumavium-intracellulare and

Mycobacte-riumfortuitum were recovered simultaneously.

Sevenspecies of mycobacteria, excluding M.

gordonae, were recovered from the 71

smear-Most Rapid Recovery Time On Conventional Media (Days)

<7 8-14 15-21 22+

<783 1 1

MostRapid Recovery Time

InBactec Medium(Days)

8-14

15-21

...

...

...

... ... ~~~~~~~~.

O~~~~~~~~~~~~~~~~~...Slower Bacte .a eTm

FIG. 2. Comparisonof recovery times for mycobacteriafrom acid-fast, smear-negativespecimens, using the BACTEC and conventional methods.

VOL.18,1983

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386 MORGAN ET AL.

Organism:

M.avium

M.tuberculosis

M.fortuitum

M.chelonei

8. 1 MEAN TIME

2 Lt ... ... ... ::.RANGE::: 35 -I BACTEC ME

I

...2...

12 _I_AI0 28

4.0

4

13.7 20.1

16L 147

26.3

3

39

6.5

4 1:;A -I8:: 22.4

] 37 18.0

.THOD

MEAN TIME

(DAYS) SAVED

BY BACTEC METHOD

18.3

Mavium 1 __: 46

M.tuberculosis 1 16 19 '2isolates 5 and 12

M.fortuitum 9 9 days longer

M.chelonei 2 __I 31

I I11.8 1 1 1 1

0 DAYS 50

Organism:

11.0 MEAN TIME

M.gastri

g.

.i

g

RANGE

+-

BACTEC

METHOD

39 46

17.0 42.5

M.kansasii

::

17'

17J35

27

13.3 31.0

M.gordonae

|91t125J,

MEAN

TIME (DAYS) SAVED BY BACTEC

38.8O

M.gastri

,3

M.kansasii 19

M.gordonae 4 l 116

I . .

....

I.

0 DAYS 50

FIG. 3. Distribution of recovery times of mycobacteria from acid-fast, smear-negative cultures, using the BACTEC and conventional methods.

negative specimens. M. avium-intracellulare

was the mostcommonly recovered (37 cultures),

followed in decreasing order ofoccurrence by

M. tuberculosis (14cultures), M.fortuitum (10

cultures), Mycobacterium chelonei (6cultures),

Mycobacterium kansasii (2 cultures), and

Myco-bacteriumgastri (2cultures).

Ofthe 71 positive mycobacteria cultures, the

conventional method detected 63 (88.7%) and

theBACTEC method detected51 (71.8%) (Fig.

1). TheBACTEC system wasfoundtobe more

rapidindetecting41 of the 43 cultures

eventual-ly detected by both systems (Fig. 2). The

re-mainingtwocultures were detected on the same

day by bothsystems.

Theranges and mean recoverytimes(in days)

for seven species of mycobacteria were

deter-mined for the BACTEC system and

convention-altechniques. The BACTEC system was more

rapid than the conventional system for the

de-tectionof all seven mycobacterial species (Fig.

3). The mean time saved bythe BACTEC

sys-tem depended on the species of mycobacteria

recovered but was more than that savedbythe

conventional system for all species evaluated

(Fig. 3and4).

The species ofmycobacteriarecovered from

the 71 acid-fast, smear-negative specimens by

conventional methods and not recoveredby the

BACTEC system were determined and

includ-ed: M. avium-intracellulare(5cultures),M.

for-tuitum (8cultures),M.chelonei(3cultures),M.

J.CLIN. MICROBIOL.

I

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TABLE 1. Comparison of media for the recovery of

mycobacteriafrom acid-fast, smear-negative

specimens(n =71)

No. of Mycobacteria recovered in:

specimens BACTEC

LU

7H10 7Hll

21 + + + +

4 + + + _

6 + + - +

5 + + - _

5 + - + +

2 + - + _

8 + - _ _

9 - + _ _

1 - + - +

2 - + + _

1 - - + +

4 - - + _

3 - - - +

No. recovered 51 44 39 37 % Recovered 71.8 62.0 54.9 52.1

tuberculosis(2 cultures), M. kansasii (1 culture), and M. gastri(1 culture). Conventional cultural methods failed to recover the following

myco-bacteria: M. avium-intracellulare (6 cultures), M.fortuitum (1 culture), and M. tuberculosis (1 culture).

In the evaluation of 71 specimens, Middle-brook 7H12 medium was more successful in recovery of mycobacteria than was any one

solid medium (Table 1). The BACTEC system

detected 71.8% of allmycobacteria, whereasLJ,

7H10, andS7H11 detected62, 55.9, and 52.1%, respectively.

Growthof M. tuberculosiswasnot significant-ly enhanced by theaddition of sodium selenate to 7H11 medium. The mean recovery times of M. tuberculosis and MOTTas determined with

Middlebrook 7H12,LJ,7H10, S7H11,and sele-nate 7H11 were analyzed. The recovery times

for M. tuberculosisandMOTTweredetermined

for thefive media. All fivemedia recovered the

mycobacteria on approximately the same day.

Therefore, studies indicated that selenate7H11

didnotdecrease the meanrecovery time of M.

tuberculosis or ofMOTT compared with other media studied.

DISCUSSION

This evaluation compared the BACTEC

sys-temwithconventionalculturing methods for the detection ofmycobacteria from acid-fast,

smear-negative, culture-positive specimens. The growth of mycobacteria from an acid-fast,

smear-negative specimen is the first clinical indi-cation of a possible mycobacterial infection.

Becauseapproximately 40% of the positive

my-cobacterial cultures inourlaboratory have

nega-tive direct acid-fast smears, the ability of the

BACTECsystem todetectmycobacteria rapidly

in thistype of specimen issignificant.

M.gordonae was eliminated from most

evalu-ations, and onlyclinically relevantisolates were

considered. This highrecovery rate ofM.

gor-donae in our laboratory, however, does present

problems with the BACTECsystem.Currently,

the BACTEC system fails to provide isolated

colonies and has no method to identify the

species of mycobacteria detected. The

suspen-sion of mycobacteria must be cultured onto

conventional media beforeabiochemical

identi-fication can be made. A culture medium was

developed containing NAP (p-nitro-a-acetyl-amino-,B-hydroxy-propiophenone), which aids in

separatingM. tuberculosis from MOTT but will

notidentifyM. gordonae(A. Laszlo and S. H.

Siddiqi, Abstr. Annu. Meet. Am. Soc.

Microbi-ol. 1982,C189, p. 303).

The BACTEC system detected 71.8% of

smear-negative mycobacteria cultures,

com-pared with 88.7% detected by the conventional

system.The latter used atotalvolume of 1.5 ml

of concentrated specimen on twoculture slants

and two agar plates, whereas the BACTEC systemusedonly0.1 ml of concentrated

speci-men to inoculate one bottle containing 2 ml of liquid 7H12 medium. The increased specimen

volume used to inoculate the fourmedia could

account for the increased detection rate

achieved by the conventionalmethod.Our

labo-ratory is presently evaluating the usage of

in-creasedinoculum size inthe Middlebrook7H12

medium to possibly increase the sensitivity of

mycobacterial detection. However, the

sensitiv-ity of the 7H12 medium for recovering

mycobac-teria exceeded that of any individual

conven-tional medium evaluated alone (Table 1). The

7H12medium recovered71.8% of the

mycobac-teria, compared with the recovery rates for LJ

(62%), 7H10(55.9%), and S7H11 (52.1%).

Sodium selenate incorporated into 7H11 agar

has beenreported toenhance the growth ofM.

tuberculosis (1).In ourlaboratory, sodium

sele-nate addition to 7H11 medium did not

signifi-cantly decrease therecoverytime ofM.

tubercu-losisorMOTTcomparedwith LJ,7H10,S7H11,

or7H12orenhance the recovery rate.

In this study, the BACTEC system proved

more rapid for the detection of mycobacteria than were conventional culture methods. The

detection of mycobacteria is helpful, but the

institution of chemotherapy depends on the

identification of the species of organism

recov-ered from the clinical specimen. The necessity

exists for either rapid identification or rapid

susceptibility testing. Techniques involving the

BACTEC system areavailablefor rapid

suscep-tibility testing. Results ofcollaborative studies

suggest that drugsusceptibility testing data can

VOL.18,1983 387

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388 MORGAN ET AL.

be obtained 4.2 to 6.9 days after detection of

mycobacterial growth; however,acorrelatecan

only be made by both methods forM.

tuberculo-sis susceptibility testing (Goodman et al., 21st

ICAAC, abstr. no.96).

Themeanrecoverytimeformycobacteriawas

decreased with the BACTEC system as

com-pared with theconventional system. However,

itmustbenoted that the BACTECsystemfailed

to detect 20 positive specimens, as compared

with8 missed by theconventional system. The

BACTEC system had particular problems in

detecting specimens yielding M.fortuitum and

M. avium-intracellulare. The conventional

sys-temshared the lowrecovery rate ofM.

avium-intracellulare butwas moresuccessful in

recov-J. CLIN.MICROBIOL.

eringM.fortuitum. Thereasonfor thisrecovery

discrepancyis notknown; perhaps the

antibiot-ics in the medium wereinhibitory.

Datafrom this and other studies indicate that

BACTEC offers promise as a method for the

rapid detection of mycobacterial growth. Atthis

time, however, this systemshould be used

con-currently with conventionalculturingmethods.

LITERATURE CITED

1. Jacquess, P. A., D. L. Smalley, and J. K. Duckworth. 1981. Enhanced growth ofMycobacterium tuberculosis in the presenceof selenium. Am. J.Clin.Pathol. 75:209-210. 2. Kubica, G. P. 1973. Identification of mycobacteria. Am.

Rev.Respir.Dis. 107:9-21.

3. Roberts, G. D. 1982.Mycobacteria and Nocardia, p. 365-406. InJ. A.WashingtonII(ed.), Laboratory procedures in clinical microbiology. Springer-Verlag, New York.

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