Brucellosis is one of the most common zoonotic diseases in Iran. Growth of Brucella is slow

and blood cultures of these bacteria are time-consuming via classical methods. We try to

evaluate BACTEC 9120 system capacity in order to detect of bacteremia due to Brucella spp and

to confirm isolated bacteria by PCR. Blood culture sample of 102 suspected patients evaluated

by BACTEC 9120 system. They were subcultured when the machine detected their growth; if

not; blind subcultures were performed on days 7, 14, 21 and 28. Forty-one of 102 suspected

patients showed bactermia. Isolation rate of Brucella was 40.2%. All patients were detected by

BACTEC 9120 system. All positive blood culture was detected via BACTEC 9120 and blind

subcultures. No positive blood culture bottles were missed by the system. Our data obtained by

using the BACTEC 9120 system indicates a more rapid detection of Brucella than conventional methods

امیرزرگر, علی اکبر

جعفری, سیروس

فتح اله زاده, بهرام

مالک نژاد, پرویز

پیری دوگاهه, هادی

English

ArUMS Digital Repository (سامانه اطلاعات زیست پزشکی و سلامت)

Introduction
Brucellosis is a widespread zoonosis caused by
members of the genus Brucella. The disease is
accidentally transmitted to humans during
occupational contact with infected animals or by
consumption of contaminated animal products (1).
Brucellosis remains an endemic disease in many
regions of the world, and is more prevalent in
developing countries, particularly in west parts of
Asia, India, Middle East, Southern European, and
Latin American countries. Human brucellosis is
endemic in all parts of Iran. The numbers of patients
recorded in 1988 were 71, 051 (132.4 per 100, 000)
(2). The laboratory confirmation of human
brucellosis is based either on isolation of Brucella
from blood cultures or detection of specific
antibodies. Blood cultures which provide the best
yield for microbiological diagnosis, with sensitivity
ranging from 53 to 90%, depend on the disease stage,
Brucella species, culture medium, quantity of
circulating bacteria, and employed culture technique.
Serology plays a major role in cases where the disease
cannot be detected by culture. Serological tests are
rapid and easy to perform (3), but their interpretation
is often difficult, particularly during the early stage of
the disease (4). Despite recent developed techniques,
like nucleic acid probes, PCR, and other molecular
techniques for Microbiological diagnosis, blood
cultures still remain the most practical and reliable
method in the diagnosis of bloodstream infections
(5). In endemic areas culture is required for a
definitive diagnosis because the symptoms of
brucellosis are non-specific, and the interpretation of
agglutinating antibody titer can be confounded by
Diagnosis of brucellosis by use of BACTEC blood culture and
confirmation by PCR
Maleknejad, P.1*,    Peeri-DoGaheh, H. 2,    AmirZargar, A. A. 3,    Jafari, S.4,    Fatollahzadeh, B.1
1Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran-Iran.
2School of Medicine, Ardebil University of Medical Sciences, Ardebil-Iran. 
3Department of Immunology, School of Medicine, Tehran University of Medical Sciences Tehran-Iran.
4Department of Infectious Diseases, School of Medicine, Tehran University of Medical Sciences,
Tehran-Iran. 
(Received  14  July  2004  ,  Accepted  9  May  2005)
Abstract:Brucellosis is one of the most common zoonotic diseases in Iran. Growth of Brucella is slow
and blood cultures of these bacteria are time-consuming via classical methods. We try to
evaluate BACTEC 9120 system capacity in order to detect of bacteremia due to Brucella spp and
to confirm isolated bacteria by PCR. Blood culture sample of 102 suspected patients evaluated
by BACTEC 9120 system. They were subcultured when the machine detected their growth; if
not; blind subcultures were performed on days 7, 14, 21 and 28. Forty-one of 102 suspected
patients showed bactermia. Isolation rate of Brucella was 40.2%. All patients were detected by
BACTEC 9120 system. All positive blood culture was detected via BACTEC 9120 and blind
subcultures. No positive blood culture bottles were missed by the system. Our data obtained by
using the BACTEC 9120 system indicates a more rapid detection of Brucella than conventional
methods. 
Key words: brucellosis, BACTEC system, blood culture. 
*Corresponding author's email:  maleknej@sina. tums. ac.
ir, Tel: 021- 88955810, Fax: 021-88953001
Code 1319
J.Vet.Res. 62,4:83-86,2007
Maleknejad, P.84
persistently elevated titer in persons without active
disease who have been repeatedly exposed to infected
animals (6). Before detection of growth, blood
culture which is done by the classical Castaneda
method can take up to 35 days of incubation (7).
Modern automatic blood culture systems have
reduced detection times of microorganisms which
produce bacteremia, allowing incubation times to be
reduced to 7 days or less (8). In this study, we aimed
to find out the rate of isolation of Brucella spp in blood
by use of the BACTEC 9120 system. 
Materials and Methods
From 2002 to 2004, 102 blood culture samples of
suspected patients were admitted to microbiology
laboratory of Tehran University of Medical Sciences.
Suspicions of brucellosis were serologically
confirmed with Rose Bengal antigen test. All the
patients were adults, and had high titer anti-Brucella
antibodies at the time of diagnosis. High titer was
defined as a titer of |||“| 1/160 by the standard tube
agglutination (STA) method. Only one blood culture
was processed per patient. Blood for culture was
drawn by the staff physicians, and between 5 to 7 ml
of blood from patients was inoculated in each of
standard aerobic/F BACTEC bottle at the patient's
bedside. Upon receipt in the microbiology
laboratory, BACTEC Aerobic/F bottles were loaded
into the instrument in the computer-assigned position
and incubated for 7 days with continuous agitation.
The BACTEC 9120 instrument was observed at 4-h
intervals for positive signals. Whenever a positive
signal occurred, the bottle was removed from the
instrument to be Gram stained and subcultured. The
BACTEC 9120 instrument monitors increases in
CO2 concentration produced by growing
microorganisms by means of a fluorescent sensor are
located in the bottom of each bottle (9). The
microscopic slid which was prepared from all
cultures showed positive and negative growth index
with gram method then they were subcultured on
chocolate agar and incubation at 37°C in
microaerophilic condition. The isolates of Gram-
negative coccobacilli were identified by
conventional tests (e. g., oxidase, catalase,
production of H2S and urease) and also by an
agglutination test using antisera. In addition, PCR
were used for precise identification of isolated
bacteria. A PCR assay with primers B4 (5'-TGG CTC
GGT TGC CAA TAT CAA-3') and B5 (5'-CGC GCT
TGC CTT TCA GGT CTG-3') (MWG-biotech,
Germany) was used to detect Brucella DNA. These
primers specifically amplify a 223-base pair
fragment from the conserved region of the gene,
which encodes an immunogenic membrane protein
of 31 KDa of Brucella abortus, specific to the
Brucella genus and present in all its biovars (Figure
1). At the end of the first week, bottles with negative
growth index were kept for an additional 3 weeks and
blind subcultures of samples from the blood culture
bottles were performed on days 7, 14, 21, and 28.
Cultures were considered negative for Brucella only
after four weeks of incubation. 
Results 
A total of 102 blood culture bottles were
processed during the evaluation period: 41 of them
(40.2%) gave a growth index positive (GIP) by the
BACTEC system and no growth was detected by
BACTEC system in 61 (59.8%) cases, and they were
growth index negative (GIN). All blood culture
bottles were subcultured on sheep blood agar,
chocolate agar and MacConkey plates. Seeded media
were incubated at 35°C in a 5% CO2-enriched
atmosphere and examined daily for 4 days. Gram
Figure 1:  Agarose gel electrophoresis and ethidium bromide staining.
Lane MW, DNA ladder; lane 1, 2, DNAs from two Brucella isolates;
Lane 3, positive control (B. abortus DNA); lanes 4, Negative control.
Diagnosis of brucellosis by use of... 85
staining was performed in all BACTEC broths.
Subculture of 41 (GIP) BACTEC broths on sheep
blood and chocolate blood agar eventually yield
Brucella organisms within two to three days. Thirty-
nine BACTEC broths gave a visible colony within
two days, but two isolates gave a visible colony after
three days. All cultures positive with the BACTEC
instrument were detected within a week and blind
subculture of 61 (GIN) BACTEC broths on days 7,
14, 21 and 28 was negative. All bottles of positive
blood culture were detected by the BACTEC 9120
instrument and by blind subcultures. Gram staining
detected organisms in 26 of 41 (GIP) BACTEC
broths. None of 61 (GIN) BACTEC broths for
Brucella showed presence of Gram negative
coccobacilli. 
Discussion 
In the last decade, automated blood culture
system, based on continuous monitoring and
colorimetric detection of CO2 production of growing
organisms has been introduced into clinical practice.
Recent studies have demonstrated that this
technology enables most clinical microbiology
laboratories to detect genus Brucellawithin the 7-day
routine incubation period (10). Yagupsky isolated 21
of 27 (78%) B. melitensis strains by using BACTEC
NR 660 system and also by blind subcultures in less
than 7 days (1). Ruiz et al., recovered 16 out of 17
(94%) isolates with BACTEC 9120 system (Becton
Dickinson) during 7 days of incubation (8). Two
different studies using BACTEC 9240 system
reported that 93 and 100% of the isolates were
recovered during 5 days (7, 10). No data on the
performance of BACTEC blood culture system for
detection of Brucella spp have been published from
Iran and this is the first and the largest reported study.
In our study, the isolation rate of blood cultures was
40.2% and in comparison with other studies (7, 10) it
seemed to be a low rate. There are three possible
explanations for the low efficiency in isolating
Brucella from our blood culture. These factors which
may be involved are one, or any combination of
factors listed below:
1- It is possible that some of the suspected patients
would be in chronic stage in which detection of
Brucella is difficult. Serra and Vinas isolated
Brucella from 92.5% of blood cultures of patients in
acute phase, but only 8.3% of those from patients
were in chronic phase (11). 
2- The effect of consumption of antibiotics on
participants, the same as the effect on suspected
patients decreases the isolation rate of Brucella in
blood culture. 
3-Since seropositive status for brucellosis is
common in endemic areas, and Iran is an endemic
region for brucellosis, thus the patients might suffer
from other chronic diseases and be seropositive for
brucellosis (12). 
We believe that if we had examined specimens
from patients who had higher titers such as |"|1/320 or
convalescence titers which are four times higher than
acute phase against Brucella, the probability of
recovering Brucella would have been improved. Our
results suggest that a 7-day incubation period in the
BACTEC system is sufficient for the detection of
these slow-growing bacteria and for detection of
Brucella spp by BACTEC system, therefore
prolonged incubation time and periodic performance
of subcultures are not required. 
Yagupsky, P.(1994) Detection of Brucella melitensis
by BACTEC NR660 blood culture system. J. Clin.
Microbiol. 32: 1899-1901.
Refai, M.(2002) Incidence and control of brucellosis
in the Near East region. Vet. Microbiol. 90: 81-110.
Dokuzoguza, B., Ergonula, O., Baykam, N.(2005)
Characteristics of B. melitensis Versus B. Abortus
bacteraemias. J. Infect. 50: 41-45.
Vrioni, G., Gartzonika, C., Kostoula, A.(2004)
Application of a polymerase chain reaction enzyme
immunoassay in peripheral whole blood and serum
specimens for diagnosis of acute human brucellosis.
Eur. J. Clin. Microbiol. Infect. Dis. 23: 194-199.
Durmaz, G., Us, T., Aydinli, A.(2003) Optimum
detection times for bacteria and yeast species with the
BACTEC 9120 aerobic blood culture system:
evaluation for a 5-year period in a Turkish University
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Abstracts in persian language 163
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bottles and the isolator aerobic culture system for detection of bloodstream infections.

Detection of Brucellae in blood cultures.

L.(1997) Diagnosis of brucellosis by using blood cultures.

Persistence of Brucella Antibody after Successful treatment of acute brucellosis in an area of endemicity.

Rapid diagnosis of Brucella Bacteremia by Using the BACTEC 9240 System.

Vinas, M.(2004) Laboratory diagnosis of brucellosis in a rural endemic area in northeastern Spain.

Diagnosis of brucellosis by use of BACTEC blood culture and

confirmation by PCR

http://eprints.arums.ac.ir/578/1/brucella.pdf

Diagnosis of brucellosis by use of BACTEC blood culture and confirmation by PCR

Abstract

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