Brucellosis is a worldwide zoonosis caused by intracellular bacterial pathogens of the genus Brucella. Although brucellosis is rarely life-threatening, it can be a severely debilitating and disabling disease and is regaining importance because of the increasing threat of its use as a biowarfare agent and its re-emergence in areas where it was thought to be nearly eradicated.1–3 Human brucellosis is endemic in Peru with B. melitensis being the most prevalent species.4–6 Peru averages more than 1,000 cases per year,7 87% of which are located in the urban centers of Lima and Callao where consumption of contaminated animal milk products most likely accounts for most cases. 4,6
Human brucellosis is difficult to diagnose because clinical manifestations are non-specific and interpretation of classic serologic test results is complex. The Rose Bengal test has traditionally been the rapid screening test of choice.8 Ruiz-Meza and others evaluated the Rose Bengal test and found that the overall sensitivity of this test was 92.9% when compared with culture.9 The specificity of the Rose Bengal test is high in populations that are exposed to the pathogen incidentally, but can be significantly lower when used to test occupational risk groups or persons from areas where the disease is endemic.9 Serologic tests such as the serum agglutination test (SAT), which are used for confirmation of results, are also hampered by a lack of sensitivity and specificity. 10–13 Furthermore, serologic testing does not provide direct evidence for the presence of the pathogen and makes confirmatory testing troublesome.14
Definitive diagnosis requires the isolation of the organism from the patient, but is restricted by the fact that Brucella spp. are slow-growing intracellular pathogens, whose successful culture from blood decreases as the disease progresses. Previous use of antibiotics also adds to the difficulty of culturing the bacillus. As such, successful diagnosis necessitates a careful selection of the best suitable culture method and validation of its performance. 15 The sensitivity of culturing Brucella spp. from blood varies from 15% to 70% when compared with clinical evidence of infection and positive serologic results depending on the study and culture method used.7,15–17
Many methods have been developed for culturing Brucella spp., including the traditional Ruiz-Castañeda (Castañeda) method, automated systems, and yield-optimizing methods such as osmotic lysis followed by centrifugation concentration or lysis centrifugation (LC). 4,18–20 The LC technique uses lysis of erythrocytes in a citrate solution, followed by isolation of Brucella bacilli by centrifugation of the sample, which concentrates the bacilli and facilitates growth after subsequent plating. We compared the LC technique with classic Castañeda culture for diagnosis of brucellosis in an area of Peru endemic for this disease.
Clinical specimens were collected from patients with clinical symptoms of brucellosis at Hospital Daniel Alcides Carrión in Callao, Peru. Patient samples were classified as acute phase, subacute phase, and chronic phase brucellosis according to duration of the disease: < 8 weeks, 8–52 weeks, or > 52 weeks, respectively. Patients were diagnosed with relapsing infection if they had completed antimicrobial drug treatment for brucellosis within the past 12 months. Patients who were serologically positive by the Rose Bengal test and had measurable antibody titers (≥1:25) by the SAT had 10 mL of blood drawn for culture. A 5-mL aliquot of venous blood was incubated at 37°C in Castañeda biphasic medium consisting of brain heart infusion agar and broth and the broth-blood mixtures were tilted over the agar phase each day for 40 days. 18 For the LC method we used a modification of the LC procedure reported by Etemadi and others; 19 this modification involved adding 5 mL of blood to 20 mL of distilled water containing 0.3% sodium citrate. The mixture was gently agitated and centrifuged at 2,000 × g for 30 minutes. The supernatant was discarded and the pellet was inoculated onto Brucella agar plates and incubated at 37°C for 7 days. 19 Bacterial colonies were initially evaluated by Gram stain, microscopy, and colony morphology. Colonies were confirmed to be Brucella spp. by polymerase chain reaction (PCR) using primers B4 and B5 specific for the 31-kD antigen of B. abortus.21 Student’s two-tailed t-test was used for statistical comparisons.
A total of 88 patients fulfilled the entry criteria over the course of 18 months (September 2005–March 2007) and donated a blood sample for culture (Table 1). Of the samples cultured, 31 (35.2%) grew Brucella spp. using the Castañeda method and 38 (43.2%) grew Brucella spp. using the LC technique. Culturing of specimens from patients with acute and sub-acute infections was significantly more successful that those with chronic or relapsing infections: 48% and 58% versus 22% and 17%, respectively (P < 0.005). Relative to the Castañeda method, the sensitivity, specificity, and positive and negative predictive values of the LC technique were 100%, 87.8%, 81.6%, and 100%, respectively. Compared with the Castañeda method, the positivity rate of the LC technique was better at all stages of the disease. Another notable finding when comparing the two culture methods was observed in the length of time to positive culture in the samples that grew: LC cultures became positive in 3.8 ± 0.8 (mean ± SD) days and Castañeda cultures became positive in 13.6 ± 6.5 days (P < 0.001), resulting in an overall difference in the mean time to positive culture of nearly 10 days (Figure 1). This finding confirmed results of previously reported studies. 5,11 The earliest growth observed by the Castañeda method was 5 days compared with 3 days by LC.
The average SAT titer of all specimens tested was 1:414 (range = 1:25–1:6,400). Although a single serum titer ≥ 1:160 is considered consistent with brucellosis when accompanied by a compatible clinical course in a patient with a history of potential exposures, 21 26% of the patients in this study had serum titers ranging from 1:25 to 1:100. These low titers cannot solely be attributed to basal community antibody levels found in brucellosis-endemic zones because we were able to culture the bacillus from 17% (4 of 23) of samples with titers ≤ 1:100.
Although these data highlight the need for more effective confirmatory serologic or molecular benchmarks, especially in high-incidence areas, they also support the use of the LC method over the Castañeda method for the culturing of Brucella spp. from blood. Our findings suggest that the LC method is more sensitive in detecting infection in patients regardless of the stage of disease, and the shorter mean detection time observed would decrease morbidity and significantly impact the public health response to natural or intentional infection. Furthermore, the LC method is simple to perform and inexpensive, making it a viable alternative for laboratories in remote or disadvantaged areas with limited technical expertise or funding. However, because of the risk of laboratory infection, strict safety precautions should be taken when handling cultures and isolates.
Type of specimen collected* and comparison of culture techniques*
Mean detection time comparing Castañeda and lysis centrifugation (LC) culture techniques. Values above graph represent the difference in mean days to positive culture between the two methods. All samples reports 31 matched cultures, including two with undetermined stage of disease, and seven non-matched LC-positive specimens. Error bars represent SEM (P < 0.001). Number of samples is indicated below the type of sample.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 4; 10.4269/ajtmh.2009.80.625
Mean detection time comparing Castañeda and lysis centrifugation (LC) culture techniques. Values above graph represent the difference in mean days to positive culture between the two methods. All samples reports 31 matched cultures, including two with undetermined stage of disease, and seven non-matched LC-positive specimens. Error bars represent SEM (P < 0.001). Number of samples is indicated below the type of sample.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 4; 10.4269/ajtmh.2009.80.625
Mean detection time comparing Castañeda and lysis centrifugation (LC) culture techniques. Values above graph represent the difference in mean days to positive culture between the two methods. All samples reports 31 matched cultures, including two with undetermined stage of disease, and seven non-matched LC-positive specimens. Error bars represent SEM (P < 0.001). Number of samples is indicated below the type of sample.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 4; 10.4269/ajtmh.2009.80.625
Address correspondence to Benjamin J. Espinosa, Naval Medical Research Center Detachment, Lima, Peru. E-mail: benjamin.espinosa@med.navy.mil
Authors’ addresses: Benjamin J. Espinosa, David L. Blazes, and Eric R. Hall, Naval Medical Research Center Detachment, Lima, Peru. Jesús Chacaltana, Hospital Nacional Daniel Alcides Carrión, Callao, Peru. Maximilian Mulder, Department of Internal Medicine, Hennepin County Medical Center, Minneapolis, MN 55415. María Pía Franco, Department of Neurology, University of Minnesota, Minneapolis, MN 55415. Robert H. Gilman, Department of International Health, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Room W5515, Baltimore, MD 21205. Henk L. Smits, Royal Tropical Institute, Amsterdam, The Netherlands.
Disclaimer: The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, or the U.S. government. Drs. Benjamin J. Espinosa, David L. Blazes, and Eric R. Hall are U.S. military service members working at the U.S. Navy Medical Research Center Detachment in Lima, Peru. This work was prepared as part of their official duties. Title 17 U.S.C. § 105 provides that Copyright protection under this title is not available for any work of the United States Government. Title 17 U.S.C. § 101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person’s official duties.
REFERENCES
- 2
Kaufmann AF, Meltzer MI, Schmid GP, 1997. The economic impact of a bioterrorist attack: are prevention and postattack intervention programs justifiable? Emerg Infect Dis 3 :83–94.
- 3↑
Troy SB, Rickman LS, Davis CE, 2005. Brucellosis in San Diego: epidemiology and species-related differences in acute clinical presentations. Medicine (Baltimore) 84 :174–187.
- 4↑
Braun W, Kelsh J, 1954. Improved method for cultivation of Brucella from the blood. Proc Soc Exp Biol Med 85 :154–155.
- 5↑
Pappas G, Papadimitriou P, Akritidis N, Christou L, Tsianos EV, 2006. The new global map of human brucellosis. Lancet Infect Dis 6 :91–99.
- 6↑
Young E, 2000. Brucella species. Mandell G, Benett J, R. Dolin R, eds. Principles and Practice of Infectious Diseases. Fifth edition. New York: Churchill Livingstone, 2386–2393.
- 8↑
Dabdoob WA, Abdulla ZA, 2000. A panel of eight tests in the serodiagnosis and immunological evaluation of acute brucellosis. East Mediterr Health J 6 :304–312.
- 9↑
Ruiz-Mesa JD, Sanchez-Gonzalez J, Reguera JM, Martin L, Lopez-Palmero S, Colmenero JD, 2005. Rose Bengal test: diagnostic yield and use for the rapid diagnosis of human brucellosis in emergency departments in endemic areas. Clin Microbiol Infect 11 :221–225.
- 10↑
Gomez M, Nieto J, Rosa C, Geijo P, Escribano M, Munoz A, Lopez C, 2008. Evaluation of seven tests for diagnosis of human brucellosis in an area where the disease is endemic. Clin Vaccine Immunol 15 :1031–1033.
- 11↑
Kassahun J, Yimer E, Geyid A, Abebe P, Newayeselassie B, Zewedie B, Beyene M, Bekele A, 2006. Seroprevalence of brucellosis in occupationally exposed people in Addis Ababa, Ethiopia. Ethiop Med J 44 :245–252.
- 12
Swai E, Schoonman L, 2008. Human brucellosis: seroprevalence and risk factors related to high risk occupational groups in Tanga Municipality, Tanzania. Zoonoses Public Health 2008 Sep 22 [Epub ahead of print].
- 13↑
Vancelik S, Gurrrrraksin A, Ayyildiz A, 2008. Seroprevalence of human brucellosis in rural endemic areas in eastern Turkey. Trop Doct 38 :42–43.
- 14↑
Mantecon MA, Gutierrez P, del Pilar Zarzosa M, Duenas AI, Solera J, Fernandez-Lago L, Vizcaino N, Almaraz A, Bratos MA, Rodriguez Torres A, Orduna-Domingo A, 2006. Utility of an immunocapture-agglutination test and an enzyme-linked immunosorbent assay test against cytosolic proteins from Brucella melitensis B115 in the diagnosis and follow-up of human acute brucellosis. Diagn Microbiol Infect Dis 55 :27–35.
- 16
Ruiz J, Lorente I, Perez J, Simarro E, Martinez-Campos L, 1997. Diagnosis of brucellosis by using blood cultures. J Clin Microbiol 35 :2417–2418.
- 18↑
Castañeda M, 1954. A practical method for routine blood cultures in brucellosis. Proc Soc Exp Biol Med 86 :154–155.
- 19↑
Etemadi H, Raissadat A, Pickett MJ, Zafari Y, Vahedifar P, 1984. Isolation of Brucella spp. from clinical specimens. J Clin Microbiol 20 :586.
- 20↑
Mantur BG, Mangalgi SS, 2004. Evaluation of conventional Castaneda and lysis centrifugation blood culture techniques for diagnosis of human brucellosis. J Clin Microbiol 42 :4327–4328.
- 21↑
Baily GG, Krahn JB, Drasar BS, Stoker NG, 1992. Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg 95 :271–275.