Detection of Brucellae in Blood Cultures
Topics
. Classic isolation procedures
. Lysis centrifugation method
. Automated blood culture systems
A. Classic isolation procedures
Isolation of brucellae from blood and other normally sterile body fluids or tissues
remains the only irrefutable evidence of brucellosis, but detection of the organism in
clinical specimens is frequently hampered by its slow growth. Based on the experience
gained with traditional methods, incubation of blood cultures for 30 days, instead of the
routine one-week period, and performance of blind subcultures have been advocated to
maximize the recovery of these fastidious organisms (1, 2). To avoid the necessity for
making repeated subcultures onto solid media, use of a biphasic medium consisting of a
solid and a liquid phase in the same blood culture bottle (Castaneda medium) has been
recommended (1).
B. Lysis-centrifugation
In 1993, it was reported that the Isolator blood culture system (Wampole Laboratories,
Cranbury, N.J.), a non automated lysis-based method with improved recovery of
intracellular pathogens such as mycobacteria and fungi, also reduced the time to detection
of circulating brucellae to two to five days (3). Use of this method has, however, some
important drawbacks:
it is not suitable for automation, it is labor intensive and
cumbersome, it is prone to contamination and requires extensive manipulation of the
specimen and the inoculated plates and, therefore, poses a substantial risk to laboratory
personnel.
C. Automated blood culture systems
In the last decade, automated blood culture systems, based on continuous monitoring
and colorimetric detection of CO2 production by growing organisms has been introduced
into clinical practice. Recent studies have demonstrated that this technology enables
detection of Brucella melitensis within the 7-day routine incubation period used by most
clinical microbiology laboratories.
In a prospective study, conducted among febrile children in southern Israel, inoculated
Peds Plus/F blood culture bottles were monitored by the BACTEC 9240 instrument
(Becton Dickinson Diagnostic Instruments, Towson, Md.) for four consecutive weeks,
and blind subcultures of negative bottles were performed once-a-week (4). A total of
2,579 blood cultures were drawn and 42 (1.6%) were positive for Brucella melitensis.
Forty-one of the 42 (97.6%) positive cultures were detected by the BACTEC 9240
instrument within two to six days. A single positive culture was missed by the instrument
and detected by blind subculture performed on day 7. Cumulative percentage rates were
23.6%, 78.9%, 86.8%, 92.1%, and 97.4% on days 2, 3, 4, 5, and 6, respectively.
Similar results were obtained in a study conducted in Saudi Arabia among a population
of febrile children and adult patients (5). Using the Standard aerobic/F (recommended for
culturing blood of adults) and the Peds Plus (used for pediatric patients), it was
demonstrated that 92.7% of 85 B. melitensis and 12 B. abortus isolates were detected by
the BACTEC 9240 instrument within 5 days of incubation.
The performance of three blood culture systems, Hemoline performance diphasic
medium (bioMerieux, Marcy l’Etoile, France), BACTEC Plus Aerobic/F and Vital Aer
(bioMerieux) were compared in a prospective study involving 17 Spanish patients with
brucellosis (6). By using a 5-day incubation protocol, 52.9, 82.4, and 11.8% of patients
were detected by the three blood culture systems, respectively. When the protocol was
extended to 7 days, the results were 76.5, 94.1, and 47.1%, respectively, indicating that
the BACTEC system was significantly faster than the comparators (P<0.05).
The results of these three studies demonstrate that the BACTEC 9240 blood culture
system enables detection of B. melitensis within the routine 7-day incubation period.
Prolonged incubation of blood culture bottles and periodic performance of blind
subcultures are not necessary.
The sensitivity and time to detection of Brucella melitensis by the BACTEC 9240 and
the Isolator blood culture system were also compared in a prospective volume-controlled
study. Blood sample aliquots, obtained from children with suspected brucellosis, were
inoculated into a BACTEC 9240 Peds Plus bottle, and into an Isolator 1.5 Microbial Tube
(7). Overall, 122 pairs of blood cultures were obtained and 28 (23.0%) were positive by
at least one method.
The BACTEC 9240 system detected all 25 positive cultures
(sensitivity: 100.0%) and the Isolator system detected 22 positive cultures (sensitivity:
78.6%) (P<0.023). Among those 22 cultures positive by both methods, 21 (95.5%) and
15 (68.2%) were found to be positive within 3 days by the BACTEC and the Isolator
systems, respectively; 8 (36.4%) were detected at least one day earlier by the BACTEC
instrument, and the remaining 14 were detected by the two systems on the same day
(P<0.045). The BACTEC 9240 blood culture system is more sensitive than the Isolator
system for the detection of B. melitensis, and is superior in terms of time to detection of
the organism. In addition, because the system is fully
automated, it allows the
simultaneous processing of a large number of bottles. Monitoring of growth is performed
by noninvasive sampling and, therefore, the risks of contamination of media or dispersion
of aerosols is minimized, an important consideration for highly transmissible and
dangerous safety level 3 organisms such as Brucella species.
References
1.
Alton GG, Jones LM.
Bacteriological methods.
In Laboratory techniques in
brucellosis, World Health Organization, Geneva; p. 17. 1967.
2.
Moyer NP, Holcomb LA. Brucella. In Murray PR, Baron EJ, Pfaller MA, Tenover
FC, Yolken RH (ed.), Manual of clinical microbiology, 6th ed. American Society
for Microbiology, Washington, D.C. ; p. 549-555. 1995.
3.
Navas E, Guerrero A, Cobo J, Loza E. Faster isolation of Brucella spp. from blood
by Isolator compared with BACTEC NR. Diagn. Microbiol. Infect. Dis. 16:7981. 1993.
4.
Yagupsky P., Peled N, Pres J, Abu-Rashid M, Abramson O.
Rapid
detection of Brucella melitensis from blood cultures by a commercial
system.
5.
Eur. J. Clin. Microbiol. Infect. Dis. 16:605-607.1997
Bannatyne RM, Jackson MC, Memish R. Rapid diagnosis of Brucella bacteremia
by using the BACTEC 9240 system. J. Clin. Microbiol. 35:2673-2674.1997.
6.
Ruiz J, Lorente I, Perez J, Simarro E, Martinez-Campos L. Diagnosis of brucellosis
by using blood cultures. J. Clin. Microbiol. 35:2417-2418. 1997.
7.
Yagupsky P, Peled N, Press J, Abramson O, Abu-Rashid M. Comparison of
BACTEC 9240 Peds Plus medium and Isolator 1.5 Microbial Tube for detection of
Brucella melitensis from blood cultures. J. Clin. Microbiol. 35: 1382-1384. 1997.