an alternative biphasic culture system for recovery of mycobacteria

Revista de Microbiologia
(1997) 28:183-189
ISSN 0001-3714
AN ALTERNATIVE BIPHASIC CULTURE SYSTEM FOR RECOVERY OF
MYCOBACTERIA AND FOR DIFFERENTIA TION OF SPECIES OTHER THAN
M. TUBERCULOSIS COMPLEX FROM BLOOD SPECIMENS
Maria Conceiíão Martins1 *, Suely Yoko Mizuka Uekil, Maria Cecilia de Almeida Palharesf, David
Jamil Hadad , Maria Alice Silva Telles I, Anna Luiza Nunes Placco2, Lucilaine Ferrazolil,
Melissa
Curcio I, Áquila Maria Lourenço Gomes I, Moisés Palaci I
Ilnstituto Adol fo Lutz, Seção de Bacteriologia, São Paulo, SP, Brasil; 2Centro de Referência e Treinamento,
DST/AIDS, São Paulo, SP, Brasil
ABSTRACT
Mycobacteremia is an increasingly frequent cornplication of late-stage infection with
lhe Hurnan Immunodeficiency Virus (I-IIV). Several different pracedures have been used
to detect and characterize mycobacteria in blood. I-Iowever, most of these are expensive
and time consuming. Our study addressed the application of an alternative biphasic blood
culture system containing modified Middlebraok 71-19 broth and Lowenstein Jensen
médium (mod 7H9/LJ) deveJoped for direct recovery of mycobacteria from blood.
Additionally, we evaluated the possibility of rapid discrimination of Mycobacterium other
than tubercle bacilli (MOTT) organisms by differential growth after simultaneous
inoculation into control media and media altered by the addition of p-nitrobenzoic acid. In
the first part of this study rnod 7H9/LJ was compared with conventional 7H9/LJ.
Mycobacterial growth curves were generated for M. tuberculosis and M. intracellulare in
both control and test medi um. r n the second part of this study rnycobacteriawere recovered
from 64 of 537 cultured specimens (11.9%). Growth was detected in 64 (l00.0%) of the
71-19/LJ, and 62 (96.9%) of the mod 7H9/LJ biphasic bottles.The mean time to
mycobacterial detection in the two systems was the sarne. For the third part of the study,
a total of \ 09\ blood specimens were cultured in mod 7H9/LJ and in mod 7H9/LJ
containing 500 ug/rnl of p-nitrobenzoic acid. A total of 72% of ali Mvcobacterium avium
complex (MAC) isolated from blood were presumptively identified correctly as MOTT
within 27 days. This study indicates that the alternative biphasic culture system has great
potential for use under laboratory conditions that prevail in developing countries.
Key words: Mycobacteria, blood, detection, mycobacterernia diagnosis
INTRODUCTION
Though historically it has been possible to recover
M. tuberculosis from the blood of patients with miliary
disease or during the initial dissemination phase of
*
tuberculous infection, until the advent of the H1V
pandemic, mycobacteremia was rarely detected (1,4).
The frequency of both silent and symptomatic
mycobacteremia
in Acquired Immunodeficiency
Syndrome (AIDS) patients and the range of species
Corresponding author. Mailing address: Instituto Adolfo Lutz, Seção de Bacteriologia,
São Paulo, SP, Brasil
Av. Dr. Arnaldo, ~51, 9º andar, CEP 01246-902,
M.C. Martins
et ai.
encountered makes the culture of blood and bone
marrow for mycobacteria an essential diagnostic tool
for the modem laboratory (2.3.4,12).
The clinical
demand for the diagnosis
of
disseminated mycobacterial disease in AIOS has led
to the developrnent of a number of comrnercial and
non-cornmercial systems aimed at the detection of
mycobacteria in sterile body fluids including Iysis
centrifugation, radiornetric broth culture, and nucleic
acid amplification and detection (1,10,16,17,20,21).
Though these tools have proven sensitive and
clinically useful, they are sometimes cumbersome or
time-consuming and moreover, are toa expensive to
be used routinely by laboratories in the developing
worId. We sought to develop a simplified altemative
method that would avoid practical
problems
associated with other systems and that would be easily
affordable. This report describes lhe laboratory and
di nical evaluation of a biphasic system containing
modified
Middlebrook
7H9
bro th
anel
Lowenstein-Jensen medium.
The study was carried out in three phases: 1) lhe
laboratory evaluation of an altemative inexpensive
enrichment method for Middlebrook 7H9 broth, 2) a
clinical study comparing the sensitivity of detection of
two biphasic culture systerns, and 3) the evaluation of
a method for direct species differentiation
of
mycobacteria growing from blood cultures.
MA TERIAL AND METHODS
Laboratory Evaluatíon of Modified Middlebrook
Media. Middlebrook 7H9 broth medium was enriched
according to a standard protocol by the addition of
ADC - bovine albumin fraction V, glucose and
catalase (Difco Laboratories, Detroit, Michigan,
USA) 10% by volume. A modified Middlebrook 71-19
broth was prepared by replacing the Middlebrook
ADC enrichment with bovine serum (heat inactivated
for 30 minutes at 56°C) to a final concentration of 10%
and p H 7,2. The ability of the two medium
preparations to support mycobacterial growth was
compared by inoculation with pedigreed strains anel
serial quantitative subculture. Standard inocula of M.
tuberculosis 1-137Rv (ATCC
25177)
and M.
intracellulare (ATCC \3950) were prepared in the
following manner. Each strain was harvested in
log-phase
growth
from 15 day cultures
on
Lowenstein-Jensen
slants and placed in a tube
containing 5 mJ of Middlebrook 71-19broth and glass
beads. A homogenous suspension was prepared by
184
shaking in a Vortex mixer for 3 minutes and the
resulting suspension was allowed to sit while larger
c1umps settled to the bottom of the tube. The
supematant was then placed in a second tube and left
undisturbed for 15 minutes to allow further settling 01'
clumped organisms. The supematant fram this smooth
suspension was then transferred to another tube anel
adjusted to a density equal to a 0,1 nm by the addition
of 7H9 broth using a nephelometer. M. tuberculosis
and M. intracellulare standardized inocula (0,5 1111
each) were added to culture tubes containing 4,5 1111
of
standard or modified Middlebrook 7H9 broth. The
capped tubes were incubated at 37°C and growth
curves were constructed by performing quantitativo
culture of the test media on Lowenstein-Jensen sianrs
at days 0,3,6,9,
and 12 after inoculation.
Clinical Evaluation
of Modified Middlebrook
Media. Biphasic culture systems were prepared in
g l as s bottles
of 50 ml capacity
using
Lowenstein-Jensen (U) medium for the solicl phase
and either standard or modified Middlebrook 71-19
broth for the liquid phase. The U mediurn was
inspissated as an 8 ml slant and a 20 mJ volume of
enriched 71-19broth was added that left roughly half of
the LJ slant submerged and half exposed. The biphasic
system using standard Middlebrook 7H9 broth with
ADC (71-19/LJ) was used as control in a cornparison
with an identical system replacing the modi·fied 71-19
broth described above (mod7H9/LJ).
Both broth
preparations were supplemented with 0.025% sodium
polyanetholsulfonate
to prevent coagulation. anel
an li bi oti cs (20 ug/rnl tri methopri m, 50 ug/ml
carbenicillin, 10 ug/ml amphotericin B) to preveni
contamination.
A total of 537 blood specimens were obtained from
patients with AIDS with suspected disserninated
mycobacterial infection hospitalized at the AIOS
Training and Reference Center (CRT A) of São Paulo.
Brazil, fromJanuary 1991 toJuly 1993. Ten milliliters
of blood collected
by venipuncture
without
anticoagulant after ioelophor disinfection of the skin
was divided into two 5 ml aliquots and immediately
inoculated
into the control
(71-19) anel te st ,
(mod71-19/LJ) media. The cultures were incubateel
upright at 37°C anel observed daily for growth for up
to 8 weeks. Bottles were inverted daily during the first
week for reinoculation of the exposed solid media.
Positive cultures were those demonstrating visible
growth in either the solid or liquid meelium, confirrned
to be mycobacterial by Ziehl-Neelsen (Z-N) staining.
Broth from the cultures remaining negative after 8
CI
Mycobactcria
weeks of incubatian was Z-N stained and examined
for acid-fast bacilli (AFB) before being discarded to
rule out growth not discovered by visual inspection.
Evaluation
of a System for Direct Mycobacterial
Species Differentiation.
A total of 1091 blood
specimens were obtained from AIOS patients with
suspicion of disserninated
mycobacterial
infection
hospitalized at the CRTA during the period from
Seplember 1993 to April 1995. Ten milliliters ofblood
callected by venipuncture without anticoagulant after
iodophor disinfection ofthe skin was divided into two
5 rnl aliquots for inoculation into each oftwo biphasic
mod 7H9/LJ médium
bottles prepared as detailed
above. One of lhe bottles also contai ned 500 ug/ml of
p-nitrobenzoi
c acid (PNB). The cultures
were
incubated upright at 3rc and inspected for growth
daily for the first two weeks, at which time they were
inverted for reinoculation
of the expcsed
solid
medium. and then weekly for an additional 6 weeks
without further inversion. Bacterial growth detected
b y visual
i n s pe ct io n was confirmed
to be
myc obact er i al or contaminant
on lhe basis of
microscopic
exami narion or stained preparations
using lhe Z-N method. For all positive cultures,
mycobacterial
growth was cornpared in the paired
írom blood xpccimcn-
bottles and cultures growing in mediurn containing
PNB were presumptively classified as MOTT.
ldentification
of Mycobacterial
lsolates.
M
tuberculosis isolates were identi fied on the basis of
p-nitrobenzoic
acicl
susceptibility.
2-thiophenecarboxyJic
acid hydrazide
resistance,
niacin production,
nitrate reduction
and colony
morphology.
(5,11) MOTT were identified at lhe
species levei using standard methods.
Statistical Methods. The cletection sensitivity of the
two biphasic systems was cornpared using the Kappa
agreernent coefficient (8).
RESULTS
This stucly was carriecl out in three phases. ln the
first phase a modified
Micldlebrook
7H9 broth
enrichment method was tested, replacing with bovine
ser um the more expensive
Mielellebrook
ADC
enr ichment.
Mycobacterial
growth curves were
generateel for M. tuberculosis anel Miintracellulare in
both control
and test médium
by quantitative
subculture at 3-day intervals. As shown in Fig. I.
similar logarithmic growth (7) was seen in both media .
. - -.- - -7H9 - M.tuberculosis
CFUlmI
- - -.- - - Mod 7H9 - M.tuberculosis
o
3
6
9
•••
7H9 - M.intracellulare
•
Mod 7H9 - M.intracellulare
12
TIME (days)
~'~ure I - l.ogarithmic growth orM.
IlIheTCII/o.\"i.\·
and M. intraccllnlnrc
in Middlebrook 7H9 broth and Modificd Middlebrook 7H9mcdiulll.
,
I
185
s::q
M.C. Martins et ai.
In second phase of the study, 537 blood cu/tures
were performed using a biphasic medium prepared
with Lowenstein-Jensen
and Middlebrook 7H9 broth.
The cultures were performed in duplicate to compare
mycobacterial
recovery and contamination
rates in
bottles with standard ADC-enriched broth 7H9/LJ) to
those of bottles with the modified broth described
above (mod 7H9/LJ). Mycobacteria
were recovered
frorn 64 of the 537 cultures (11.9%), 64 of which
(IOO.O%) were detected by the 7H9/U bottles and 62
(96.9%) by the mod 7H9/LJ bottles. Kappa coefficient
calculation
showed excellent
agreernent (91.0%)
between systems.
Of lhe 64 isolates, 37 (57.8%) were MAC and 24
(37.5%) were M. tuberculosis cornplex organisms,
Three isolates could not be identified at the species
levei because of contaminating overgrowth or laek of
growth on subculture.
Primary
contamination
occurred in I I (2.0%) and 8 (1.5%) ofthe 7H9/LJ and
1110d7H9/LJ biphasic systems, respectively (Table I).
The number of days required for detection of
positive eultures did not differ between the two
systems (Table 2). Myeobacterial growth was detected
by inspection ofboth the liquid and solid phases ofthe
culture systern.
In the final phase of the study a method of species
differentiation
directly from the inoculated culture
Tahle
I - Rccovcry
01"rnycobacteria
spccimcns
in two biphasic
~:nhlood
Biphasic
Positive cultures
n=64
systcrn
MAC
MT
.'i
I
o
2
2
O
12
)'
7H9/LJ"
h
Mod.7H9/LJ
7H9/1J
Mod.
and
7H9/L.I
a
Middlebrook
h
Modificd
and conramination
culturc systcrns
7H9 broth
Middlcbrook
MAC - Mycobacterium
MT - Mycobactcrium
NI
rales
Contaminatcd
cultures
n=12
4
-.'
7
and Lowcnstciu
7H9 broth
Jcnscn
and Lowcnstcin
Jcnscn
avium cornplcx
ruberculosis
cornplcx
NI - not idcnti licd spccics
Tahle
2 - Number
01"days rcquired
Organisrn
N°
for dctection
h
" Excludin!!
h
10 isolares
32
that could
Mycohoc;crillln avium complcx
186
not bc identified
The well-documentecl
association of AIOS with
Disserninated Mycobacterial
Infection prornpted the
investigation
of methods
for the detection
and
identification
of these organisms
from blood
specimens.
Although biphasic blood cultures are
commonly used for lhe isolation of bacteria anel fungi.
they are seldom used for the isolation of mycobacteria
The design of our study addressed the application of
an alternative blood culture system for diagnosis and
for monitoring
the efficaey of therapy under lhe
prevailing
laboratory
eonditions
in developing
eountries.
In this phase of our study, the simple replacement
of ADC supplement with bovine serum to a final
concentration of 10% in Middlebrook 71-19 médium
did no! affect the growth of M. tuberculosis or M.
intracellulare (Fi g , I). This may be a useful
substitution for containing eosts (9), although, as with
any other in-house media supplemented with serum or
blood, factors may be present that possibly affect
({I'il/I/I
complcx
organisms.
Mod 7H9/
7H9 / L.I
1...1
Mcan
Range
~J.:\
18-6()
~ 1.:1
17-60
27.1
14-60
27.2
15-60
Mcan
MAC
DISCUSSION
of lhe M. tubcrculosis and M.
or isolares"
M.{l/hel"C/({osis
Irom
medium was evaluated. Pairs of biphasic 7H9/L.J
bottles, one containing PNB, a speci fie inhibitor of M.
tuberculosis, were inoculated with 5 ml ofblood frorn
AroS patients
with suspicion
of disserninatert
mycobacterial infection. Of 1091 cultures, 58 (5.3%)
were positive for mycobacteria.
Of the 43 isolates
given a final idenfication of MAC, 31 (72.1 %) eoulel
be presurnptively identified as MOTT over an average
of 27 days on the basis of growth
in t hc
PNB-containing
bottle. The 12 MAC isolates that
failed to grow in the PNB-containing systerns were ali
subsequently
shown
to be PNB-resistant.
Misidentification
of M. tuberculosis did not occur anel
ali of these 15 isolates were growth inhibitecl by the
selective biphasie bottle (Table 3).
Five isolates could not be identfiecl at the species
leveI because of either contaminating overgrowth 01'
lack of growth on subculture. Primary contamination
occurred in 12 (2.23%) biphasic systems.
Range
Mycobaclcria
'fable
for MOTT in 1091 blood specimcns dircctly
inoculatcd into lhe mod 7H9/LJ biphasic systcm
anel into lhe 7H9/IJ
biphasic sysicm conlaining
PNFl
3 - Scrcening
anelsilllultancollslv
(canlrol)
(PN81
PN8 (+)
Idenli [ication
Control
(+)
M. tuberculosis
O
MAC
29
Mod 7H9/LJ
- Modificd
biphasic systcm
PNB - -nitrobcnzoic
PNB (-)
Control
r-)
Conlrol
(-)
TOlal
(+)
1'1
1076
1091
12
104~
1091
Control
O
2
Middlebrook
7H9 / Lowcnstcin
Icnscn
acid
(+) mycobaclerial
growth
(_) abscucc of mycobactcrial
growth
MOTT - Mycobacrerium othcr Ihan tubcrclc
MAC - Mvcobactcrintn aviuin complcx
hacilli
bacterial growth ( 14). Several methods have been used
to recover mycobacteria
frorn blood cultures. For
example. Agy et al. (I), using the isolator systern,
recovcred
mycobacteria
from 29 of 180 blood
specimens submitted to culture (16.1 %). Salfinger et
al. ( 16) detected mycobacteria in 17.6% of the blood
samples examined
using sodiurn deoxycholate
solution-lysis-centrifugation
as the concentration
technique, followed by inoculation of the sediments
into radiornetric BACTEC (7HI3) (14). Strand et al.
(18) reported that BACTEC 13A médium revealed the
presence of mycobacteria in 63 of 1848 blood culture
(3.4%). ln the present study carried out for médium
I
I evaluation with clinical sarnples, mycobacteria were
recovered from 64 of the 537 cultures (12.1 %),64 of
: which (100,0%) were detected with the 7H9/LJ bottles
and 62 (96.9%) with the mod 7H9/LJ bottles. It should
be noted also that our biphasic médium contained
srnaller amounts of liquid medi um than usually
recomrnended (3,6, \1).
It is important to emphasize that factors outside the
laboratory
such as therapeutícs
and clínician
indications for mycobacterial
cultures may greatly
influence lhe rate of recovery of mycobacteria frorn
blood in these studies (1,4).
W it h respect to the ti me needed to detect
mycobacterial
growth, there was no significant
difference between the biphasic systems studied. The
wide range of growth detection times observed was
probably due to the variable nurnber of bacilli in the
bJooel speci mens. As demonstrated by Ber! in et al. (3),
lhe time of recovery is inversely proportional to the
number of organisms inoculated.
The ability of mycobacteria to grow 011 media
containing differentially
inhibitory substances has
I
I
Irom blood spccirnens
repeatedly been shown to be of value for ielentification
purposes (13,19).
The M tuberculosis complex is inhibited by PNB
at a concentration of 500 g/ml, whereas MOTT are
resistant to this concentration.
The latter, however,
may present variation in sensitivity, with the existence
of a small percentage of strains sensitive to certain
drug concentrations, as proposeel by Rastogi et al, (15)
and Tsukamura et ai. (19). ln the present study. after a
rnean growth of 27 days in medium containing PNB.
31 strains (72.1 %) were considered to be MOTT. This
means that the laboratory can supply a presumptive
MOTT result on the basis ofthe growth ofthese strains
on medium containing PNB. Ali of these strains were
later confirmed to be MAC on the basis of the results
of biochemical tests.
With respect to cultures presenting growth only on
control rnedium, i.e., strains sensitive to PNB, it was
not possible to provide a presumptive result since 15
(100%) were identified as M tuberculosis and 12
(27.9%) as MAC. One of the tests used for MOTT
identification is resistance of the strain to 500 gim]
PNB. Resistance
to this PNB concentration
was
observed
inl2
MAC st r a i n s during
their
identification,
suggesting
that the bacillary load
inoculated into the two flasks (contraI medium anel
medium modo 7J9/LJ with PNB) was so low that it was
inhibited by this drug con~entration.
Two strains
that
presented
growth
in
PNB-containing
medi um did not grow on control
medium (with no PNB), suggesting that the samples
were paucibacillary,
permitting the occurrence of a
difference between the inocula added to the two
media.
For analysi s of the agreement between methods for
the presumptive identification ofMOTT by growth on
medium 7H9/LJ containing
PNB and by MAC
identification using biochemical methods, the Kappa
coefficient was calculated with a 95% confidence
interval, giving a value of 0.799 (0.696-0.90\).
This
value may be considered a measure of the relative
efficacy of the method of presumptive identification
since it anticipates the definitive results obtained by
the biochemical tests with reasonable concordance.
The differentiating
ability of PNB-containing
biphasic media appears to be sufficiently rapid and
specific to be useful in a clinical laboratory, since
identi fication of mycobacteria
by conventional
methods may take up to I month.
In conclusion, the alternative biphasic systern is
compact,
self-contained,
simple
to use and
I f)7
M.C. Martins
e! al.
inexpensive. Ali of these advantages make it suitable
for use under the prevailing laboratory conditions of
developing countries.
ACKNOWLEDGMENTS
We thank Dr. Mark Perkins for valuable
suggestions and Dr. Jose Leopoldo Ferreira Antunes
for statistical analysis.
aplicabilidade na rotina diagnóstica
desenvolvimento.
Palavras-chave:
Micobactéria,
diagnóstico, micobacteremia
I.
2.
Micobacteremia constitui uma das manifestações
oportunísticas mais frequentes dos estágios avançados
da infecção pelo virus da imunodeficiência humana.
Diversas metodologias tem sido propostas para o seu
diagnóstico, no entanto, a maioria destas apresentam
custo elevado ou complexidade de operação técnica.
Baseados nestes fatos nos propusemos a avaliar um
sistema alternativo de cultura bifásico contendo
Middlebrook 7H9 modificado Lowenstein Jensen
(rnod 7H9/L.n. especificamente adaptados para (i) o
isolamento de micobacterias, (ii) triagem de outras
espécies que não as do complexo Mycobacterium
tuberculosis (MOTT) através da adição de ácido
p-nitrobenzoico aos meios (mod 7H9/LJ+PNB). Para
a primeira etapa do estudo comparou-se os meios mod
7H9/LJ e 7H9/LJ convencional, realizando-se a curva
de crescimento das espécies M. tuberculosis e M.
intracellulare em ambos os meios. Na segunda etapa
do estudo isolou-se
um total de 64 cepas de
micobacterias a partir de 537 espécimes de sangue
(11.9%), das quais 64 000,0%) em 7H9/LJ e 62
(96,9%) em mod 7H9/LJ. Verificou-se um tempo
semelhante de detecção destas micobacterias em
ambos os sistemas. Para a terceira etapa do estudo,
cultivou-se um total de 1091 espécimes de sangue nos
sistemas mod 7H9/LJ e 7H9 mod/LJ contendo 500
ug/rnl de ácido p-nitrobenzoico, constatando-se que
um total de 72% dos organismos
isolados e
pertencentes ao complexo Mycobacterium avium
puderam ser presuntivamente
identificados como
MOTI em 27 dias. Estes resultados, associados a
simplicidade e baixo custo destes sistemas bifásicos
constituem
elementos
potenciais
para sua
Illg
sangue,
detecção.
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