Introduction

Brucella species are the etiological agents of brucellosis. Five species have been recognized in the past, according to relative animal host specificity: B. melitensis in sheep and goats, B. abortus in cattle, B. suis in swine, B. ovis in sheep, B. canis in dogs, and B. neotomae in desert rats [1]. More recently, marine mammals have been recognized as an additional animal reservoir for Brucella species, and B. cetaceae and B. pinnipediae are the newly proposed species names associated with cetaceae and pinnipediae, respectively [2, 3].

Diagnosis of human brucellosis remains based upon the isolation of Brucella sp. from the blood or other clinical samples of infected patients [4]. However, the sensitivity of culture for detecting Brucella is low in patients with subacute or chronic disease, or when an antibiotic therapy has been administered before clinical samples have been collected for Brucella culture [4]. Serological techniques are the usual alternative diagnostic methods used when cultures remain negative [5] and, among these, the Wright test is still considered the standard method. The sensitivity of serological tests varies from 65% to 95% [5]. However, low specificity is the major limitation of serological techniques due to serological cross-reactions [5], especially between Brucella spp. and Yersinia enterocolitica O:9, but also, to a lesser extent, Francisella tularensis, Vibrio cholerae O:1 (especially in patients vaccinated against cholera), E. coli O:157, Salmonella O:30, Afipia clevelandensis, Ochrobactrum anthropi. Thus, PCR-based [6–11] and real-time PCR (rtPCR)-based diagnostic methods [12–15] have been developed in the last two decades to detect Brucella DNA in human samples. PCR has proven to be more sensitive than culture in patients with focalized brucellosis [9], and it is particularly useful when an antibiotic therapy has been administered before clinical specimen collection for Brucella culture [9]. PCR is also less hazardous than culture for laboratory workers [7]. Our goal was to evaluate a new rtPCR assay for detection of Brucella DNA in human serum samples from brucellosis case patients.

Materials and methods

Reference strains and human isolates of Brucella and non-Brucella species used in the present study are listed in Table 1. Brucella spp. were grown on Columbia agar plates supplemented with 5% sheep blood (bioMérieux, Marcy l’Etoile, France) in a biosafety level 3-equipped laboratory. DNA extraction from pure cultures of Brucella and non-Brucella strains was performed using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). DNA concentration and purity was measured using an absorbance ratio between 260 and 280 nm.

Table 1 Brucella and non-Brucella strains used in the present study
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rtPCR was performed using the Light Cycler instrument (Roche Diagnostics, Mannheim, Germany). The target DNA was a 169-bp portion of the bcsp31 gene, encoding a 31-kDa surface protein that is found in all Brucella species and biovars [7]. Primers and probes were as follows: forward primer BCSP31fw [5’-TCA ATG CGA TCA AGT CGG-3’]; reverse primer BCSP31rv [5’-GCA TCC TTA CGC GCA A-3’]; hybridization probes 5’-ACG CAG TCA GAC GTT GC-(fluorescein)-3’ and 5’-(LCRed 640)-ATT GGG CCT ATA ACG GCA CC-(P)-3’. Each reaction mixture contained 10 pMol of each primer, 4 pMol of each hybridization probe, 100-nMol MgCl2, and 5 μl of DNA extract in (20 μl final volume) Fast Start Master Hybridization Probes reaction mix (Roche Diagnostics).

The PCR profile was as follows: initial template denaturation at 95°C for 8 min, and 45 cycles of template denaturation at 95°C for 10 s, primer annealing at 57°C for 10 s, and primer extension at 72°C for 8 s, with a temperature transition rate of 20°C/s for all steps. A negative control (purified PCR-grade water) and a positive control (B. melitensis 16M genomic DNA) were included in all PCR assays.

A B. melitensis 16M genomic DNA suspension was prepared in sterile distilled water and titrated spectrophotometrically. Assuming a molecular mass for the B. melitensis genome of approximately 3 fg of DNA [7, 12], this suspension contained approximately 6×105 genome copies per 5 μl DNA extract. For each PCR assay, tenfold serial dilutions (10−1 to 10−7) of this external standard were run in parallel with serum samples to be tested, and the log of the concentration of each dilution series was plotted versus the cycle number at which the fluorescent signal increased above a threshold value (Ct value). The slope of the standard curve generated allowed calculation of the reaction efficiency (e) according to the following equation: e=10−(1/slope). Intra-assay and inter-assay reproducibility of DNA titration were assessed by calculation of variation coefficients. The analytical sensitivity of the rtPCR assay was defined as the minimum B. melitensis DNA concentration that could be amplified in ≥90% of ten independent experiments.

In order to evaluate the influence of serum or blood on the efficiency and analytical sensitivity of our rtPCR assay, serum or blood samples from volunteers were spiked with the B. melitensis 16M DNA suspension and used for rtPCR testing.

As a laboratory associated with the French Reference Laboratory for Brucella at AFSSA (Agence Française de Sécurité Sanitaire des Aliments, Maisons-Alfort, France), we receive serum samples from suspected cases of human brucellosis for diagnostic expertise. We selected 17 serum samples from 17 patients with culture-proven brucellosis for this study. Control samples included 30 sera from blood donors, and sera from five patients with systemic lupus erythematosus, five patients with Epstein Barr virus infection, five patients with cytomegalovirus infection, five patients with acute Coxiella burnetii infection, five patients with Lyme disease, and five patients with Bartonella henselae (cat-scratch disease) infection. Serum samples were tested for the presence of antibodies against Brucella and Y. enterocolitica O:9 antibodies (Table 2), and for the presence of Brucella sp. DNA by rtPCR (DNA was extracted from 200-μl samples).

Table 2 Results of rtPCR and serological test for the 17 patients with brucellosis as proven by culture of a Brucella strain from blood (patients 1–14) or synovial fluid (patient 15)
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Results and discussion

Using genomic DNA extract, our rtPCR protocol amplified the expected 169 bp amplicon from the 15 Brucella strains, but not from the 42 non-Brucella species. Interestingly, O. anthropi DNA was not amplified, whereas cross-amplification has been previously reported with bcsp31-targeting PCR assays [6, 10–12]. Thus, species specificity and selectivity of our rtPCR assay were 100%. When using B. melitensis 16M DNA suspension in sterile distilled water, statistical analysis revealed high reproducibility of the standards, with intra-assay and inter-assay variances ranging from 0.0022 to 0.075 and from 0.0027 to 0.12, respectively. Reaction efficiency was 1.95.

A linear regression was found between the log of B. melitensis 16-M genome copies and Ct values (y=−3.43x+39.26, R 2=0.99), over five orders of magnitude (from 7.14×105 down to 71.4 genome copies/5 μl DNA extract). The analytical sensitivity was found to be seven genome copies (∼21 fg DNA) per 5 μl of DNA extract. When using DNA templates prepared from serum or blood samples spiked with B. melitensis 16 M, reaction efficiencies were 1.95 and 1.98, respectively. The equation for the linear regression line for the standard curves generated and its corresponding R 2 value were, respectively, y=−3.44x+35.2, R 2=0.99 and y=−3.035x+39.01, R 2=0.99. A linear regression of over five orders of magnitude was found, from 6×105 down to 60 genome copies/5-μl DNA extract. The analytical sensitivity was found to be six genome copies (∼18-fg DNA) per 5 μl of DNA extract for serum samples, but 60 genome copies (~180-fg DNA) per 5 μl of DNA extract for blood samples. Limits of detection of Brucella DNA extracted from human blood samples varying from 250 fg down to 10 fg have been reported in the literature [8, 10, 12, 15].

The 17 brucellosis patients (10 men/seven women; mean age, 46 years; age range, 11–69 years) presented with clinical manifestations compatible with acute or subacute brucellosis, e.g., asthenia (7/17), high fever (6/17), sweats (5/17), myalgia (4/17), arthritis (3/17), orchiepididymitis (2/17), spondylitis (1/17), sacroiliitis (1/17), or aortic aneurysm infection (1/17).

Species and biovar determination of isolated strains was performed at AFSSA (Table 2). Fifteen of the 17 serum samples gave a strong positive reaction in the plate agglutination test. Brucella and Y. enterocolitica O:9 antibody titers are presented in Table 2. Eleven of the 17 serum samples tested with rtPCR gave a positive result, corresponding to a sensitivity of 64.7% (95% confidence interval, 42–87.4%). Bacterial titers ranged from approximately 25 to 650 genome copies per 5 μl of DNA extract (Table 2). In the literature, sensitivities and specificities of PCR and rtPCR assays targeting the bcsp31 gene to detect Brucella DNA in human blood or serum samples have varied from 58% to 100% and from 91.9% to 96.5%, respectively [8, 10, 14]. Interestingly, most patients with a positive rtPCR test result showed high IgM antibody titers for Brucella spp. (median, 640; range, 20–2,560 versus median,<20; range,<20–160 for rtPCR-negative patients), whereas IgM-type antibodies are usually found early in the course of brucellosis [5]. The low sensitivity we found may be partly explained by inadequate preservation of many serum samples (>3 months at −20°C) before rtPCR testing. A higher sensitivity may be expected with freshly collected samples.

Serological tests to detect Brucella spp. were negative for all 30 serum samples from blood donors. Among sera from the 30 non-brucellosis patients, none tested positive with the plate agglutination test or the Wright test, while one sample from a systemic lupus erythematosus patient displayed anti-Brucella IgG and IgM titers of 160, and another sample from a Lyme disease patient displayed an IgG antibody titer of 80. The Brucella BCSP31 rtPCR test was negative for sera from all 60 control patients not infected with Brucella spp., corresponding to a specificity of 100%.

In conclusion, we report the use of a bcsp-31 gene-targeting rtPCR test that detected Brucella DNA in serum samples from bacteremic brucellosis cases. This test will allow us to confirm diagnosis in patients with suspected brucellosis but negative blood cultures. It may also serve as a rapid and safe method for identifying Brucella strains isolated from human or animal samples.