| Abstract|| |
Background: Seronegative brucellosis is occasionally encountered in clinical practice especially in localized disease where diagnosis is reached mainly through positive blood culture. Cellular immune responses are pivotal for protection against intracellular bacteria such as Brucella. This study was performed to evaluate the expression of activation markers on peripheral blood mononuclear cells in response to in vitro stimulation by whole-cell suspension of Brucella melitensis for the diagnosis of brucellosis. Materials and Methods: Fifteen seronegative patients with positive blood cultures for Brucella and twenty-five unexposed healthy blood donors serving as controls were recruited for the study. Peripheral blood mononuclear (PBMC) cells were obtained by the method of Ficoll Hypaque density gradient and stimulated in vitro with Brucella antigen. Expression of activation markers was assessed by flow cytometry after staining of PBMC with mononuclear cells with relevant monoclonal antibodies. Results: Incubation with mitogen induced expression of all the four markers was demonstrated in all blood samples. In contrast, samples from all patients of Brucellosis showed significant positive responses with the expression of activation markers (CD38, CD69, CD25, and CD71) on both CD4+ and CD8+ cells as compared with the control group (P < 0.001). Conclusion: It is inferred that there was a remarkable upregulation of activation markers on CD4+ and CD8+ in seronegative patients with Brucellosis. It is recommended that the method can be utilized as a novel diagnostic test for detection of brucellosis where serology is negative.
Keywords: Diagnosis, immune system, lymphocyte, seronegative brucellosis
|How to cite this article:|
Almajid FM. Lymphocyte activation test for diagnosis of seronegative Brucellosis in humans. Indian J Pathol Microbiol 2011;54:775-81
| Introduction|| |
Isolation of Brucella More Details organism from patients' samples remains the gold standard for the diagnosis of Brucellosis More Details. However, due to the prolonged incubation period and other confounding factors, yields are usually obtained in 15-70% of samples.  Traditionally, Brucella species are cultured in Castaneda medium with subcultures taking around four weeks to achieve a positive result.  The advent of new automated blood culture system (BACTEC 9240) offers an accelerated (usually within 7 days) and efficient (≥ 90%) diagnostic modality. , Despite the advance in diagnosis, bone marrow cultures are more sensitive than blood cultures for the diagnosis of brucellosis.  However, harvesting bone marrow for culture is an invasive, painful procedure and the results lack reproducibility. Furthermore, handling of Brucella is a risk to laboratory personnel and appropriate precautions should be taken. ,
A variety of serologic tests have been used for the diagnosis of brucellosis, of which, the serum agglutination test (SAT) remains the most popular.  A titer of 1:160 or more in a compatible clinical setting is usually considered as diagnostic. There are, however, some drawbacks in the use of SAT as there are subsets of patients in which the infection failed to stimulate the host immune response especially with the localized disease even without an overt immune deficiency. ,, Serum agglutination test has another drawback in that it may not be suitable for patient's follow-up, since titers can remain high for a prolonged period. SAT also lacks the ability to diagnose B. canis infection. ,, The Coomb's test, on the other hand, utilizes antibodies to human globulins to test for the presence of non-agglutinating antibodies in suspected patients when the standard tube agglutination is negative.  The enzyme-linked immunosorbent assay (ELISA) measures IgM, IgG, and IgA immunoglobulin which allow better interpretation of the clinical condition. The ELISA was found to be comparable and overcome some of the shortcomings of the serum agglutination test. A comparison of ELISA test with the serum agglutination test yields higher sensitivity and specificity.  Similarly in patients with neurobrucellosis, ELISA offers significant diagnostic advantages over conventional agglutination methods.  In addition, it has been reported to differentiate between active and inactive infection.  The introduction of specific polymerase chain reaction (PCR) in the recent past has facilitated detection of Brucella organism as early as 10 days. Although nested PCR has a higher specificity and sensitivity, it is, however, prone to contamination.  Real-time PCR is likely to be the diagnostic tool of the future, offering the possibility of detection within 30 minutes. ,, However, PCR tests are expensive and may not be readily available in most centers across the globe.
T-cell activation followed by proliferation is a standard method to evaluate the immune response against intracellular organisms. Infection with Brucella species results in the activation of cell-mediated immune responses. , Cellular immune response had been studied in murine models where cytokine secreting CD4 cells have been shown to be fundamental components of immunity against Brucellosis. , Many studies have utilized the measurement of tritiated thymidine ([3H]TdR) incorporation into the DNA of proliferating cells after stimulation of lymphocyte by Brucella antigen in the diagnosis of animal Brucellosis. ,,,,, However, there are only few studies which applied the [3H] thymidine uptake method in the test for active Brucellosis in humans. , This thymidine technology only gives information on overall proliferative responses without providing the specific subset of cells involved in these responses. In addition, it is time-consuming and requires special technical expertise. Activation of T-lymphocytes are known to express on the surface a number of molecules such as CD25, CD69, CD71, and HLA-DR which are usually expressed minimally or even absent on resting cells. , These are called activation markers and they are easily measured by flow cytometric analysis using monoclonal antibodies as specific reagents. It is also possible to use immunofluorescent staining to identify the markers within bulk lymphocyte cultures and the T-cell subpopulations involved in the activation process. Previous studies had reported the usefulness of CD69 expression for the rapid assessment of functional response by individual T-cell subsets to a variety of stimuli. ,, The flow cytometric assessment of the activation marker on stimulating T-Cell was shown to correlate well with the cell proliferation determined by tritiated thymidine.  Martha et al. studied the measurement of the early activation marker (CD69) and found that only patients with brucellosis responded with expression of CD69 on T-cell.  Few studies had evaluated the lymphocyte activation test in patients with active brucellosis and positive serology, but none has utilized the test in humans with active brucellosis and negative serology. , Kaneene  studied the test in infected cattle where it demonstrated significant sensitivity and specificity. To our knowledge, there is only one case report of a patient with negative serology who was diagnosed initially by lymphocyte activation test and later confirmed by blood culture. 
| Materials and Methods|| |
Fifteen patients (age 30-46) comprising 12 male and 3 female, with the diagnosis of acute brucellosis confirmed on blood cultures, with no serological evidence of infection, were recruited in this study. All the patients were diagnosed to have acute disease (duration of illness, 21-200 days, mean of 60 days) as defined by Young.  Twenty-five normal male blood donors (age 29-46) who tested negative for brucellosis by repeated standard agglutination tests and blood cultures were recruited as controls.
A total of 10 ml blood was collected by venipuncture from patients and controls. The sample was divided into seven milliliters of blood in EDTA tube (Becton Dickinson) and three milliliters of blood in a container without anti-coagulant for serum separation. The latter was kept at -20° C for serological assay. Of the seven milliliters aliquot, five milliliters of blood was used for cell separation using Ficoll Hypaque (Bio test AG UK) density-gradient centrifugation.  The remaining two milliliters sample was used for complete blood count.
Brucella antigen preparation was done utilizing live culture of Brucella melitensis Rev-1 strain, (from SYVA Laboratories, Leon, Spain) which was immediately transferred to trypticase soy agar for growth in trypticase soy broth (Becton, Dickinson, USA) supplemented with 12% yeast extract (Oxoid, Unipath, UK) and 30% glucose. Cultures were incubated at 37°C for 48 h with continuous shaking (New Brunswick rotary shaker). Inactivation of Brucella organisms was carried out by the addition of 10% formaldehyde saline to the culture in accordance with previously studied method. Brucella organism were then diluted to 10 9 CFU/ml in 0.85% saline, chilled on ice, and sonicated with a cell disrupter (Soniprep 150 MSE UK) set at 20 mm of amplitude with three minutes pulses and then 30-second pause. The sonicated bacteria were centrifuged at 20,000 x g for 30 minutes and the supernatant was collected. The antigens were assayed for the protein concentration and lipopolysaccharide by 3-deoxy-d-manno-2-octulozonic acid (KDO). , Brucella antigen was used as specific antigen and phytohemagglutinin was used as positive control.
Lymphocyte Stimulation was performed as described previously by Maino et al. Freshly isolated PBMCs were suspended in complete RPMI-1640 medium supplemented with 10% heat-inactivated bovine serum (Imperial, UK) 2 Mm L-glutamine (Life Technologies, USA), 100 IU/ml penicillin, and 100 μg/ml of streptomycin (Life Technologies, UK). PBMCs were separated into three Falcon tubes, each containing 2×10 6 cells/ml. To one tube, 10 μg/ml PHA was added, whereas to another tube, 10 μg/ml of Brucella antigen was added, and in the third tube, 1 ml RPMI-1640 was dispensed in PBMCs serving as a control. All tubes with their contents were incubated at 37 o C in 5% CO 2 in a humidified incubator (Forma Scientific) for 72 hours.
Direct Immunofluorescence Staining of PBMCs
After incubation, cell cultures were transferred to 15 ml conical tubes and washed twice with wash buffer (500 ml of sterile RBMI-1640 with L-2mm glutamine and 10 ml [×100] Hepes buffer). Cells were suspended in wash buffer and the concentration of cells was adjusted to 2×107 cells/ml. Two sets of four tubes were then prepared for immunofluorescent staining. To one set of four tubes, 20 μl of anti-CD4-PE, and to the other, 20 μl of anti-CD8-PE monoclonal antibodies were added. An aliquot of 40 μl of the cell suspension was added to each tube. Twenty microliters of fluorchrome-conjugated monoclonal antibodies against activation markers (CD69-FITC, CD25-FITC, CD38-FITC, and CD71-FITC) were added to the relevant tube in parallel for both CD4 and CD8 set of tubes. The tubes were incubated for 20 minutes on ice. Two milliliters of cold wash buffer was added to each cell suspension and the samples were centrifuged at 300 ×g for 5 min at 4°C. The supernatant was discarded and the cells were resuspended in 0.5 ml of 1% paraformaldehyde and kept at 4°C until analyzed.
Flow cytometric analysis was performed on the FACScan (Becton Dickinson Immuno-cytometry, San Jose, CA) and 3x10 4 events were collected for each sample. Analysis was performed using CELL Quest software package (BDIS) on list mode data and the lymphocyte gate as defined by forward/side scatter characteristics. For two-color analysis, FL1/FL2 contour plots were employed, whereas for all experimental conditions, matched subclass controls were employed to determine the level of auto fluorescence and nonspecific binding. Activation antigen expression was determined on two-color contours by setting quadrants using the appropriated conjugated subclass controls. In order to evaluate changes from the baseline (CD69, CD25, CD38 and CD71), percentage of both CD4 + and CD8 + expressing activation markers before and after stimulation were obtained by data analysis using Cell Quest software package (BDIS).
Data were analyzed using the Statistical Package for the Social Sciences (SPSS) V-10 statistical software. The means and standard deviations (SD) were calculated for each marker. The student's t- test was used to compare T-cells subsets and activation markers between population groups. A P value of < 0.05 was considered significant.
| Results|| |
All of the 15 patients had negative standard tube agglutination test (STA) repeated at a two-week interval. They were also negative tests with 2-mercaptoethanol and Coomb's test. Active Brucellosis was however confirmed in all patients by positive blood culture. The mean percentage values of T-cells (both CD4 + and CD8 + ) obtained from the 25 healthy individuals expressing each activation markers (CD69, CD25, CD71, and CD38) before and after PHA stimulation is shown in [Table 1] and [Figure 1] and [Figure 2]. The CD38 marker gave the highest value (mean 37.8% and 29.2%), followed by CD69 (mean 35.6% and 28.7%) and both were significantly higher than that of cells positive for CD25 (21.4% and 22.8%) and CD71 (21.3% and 21.8%) from both CD4+ cells and CD8+ cells, respectively. The kinetic expression of these four activation markers from both CD4+ and CD8+ cells for the control group is presented in [Figure 3] and [Figure 4]. The mean percentage of the CD4+ and CD8+ cells expressing the activation marker was plotted at 24 h, 48 h, 72 h, and 96 h. The measurement which was started at 24 h showed CD38 having the highest value, peaking at 48 h and declining though still at significant level at 96 h. The expression of CD69 followed closely peaking at 72 h, while both CD71 and CD25 induced a peak of expression between day 2 and day 3 on both cells. The pattern was then followed by a gradual decline in the expression of all markers as measured at day 4 .The un-stimulated sample did not show any expression of the activation markers tested for both cells.
|Figure 1: Expression activation markers on CD4+ Cells from healthy control before and after stimulation with PHA mitogen (n=25)|
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|Figure 2: Expression activation markers on CD8+ Cells in healthy control before and after stimulation with PHA mitogen (n=25)|
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|Figure 3: Kinetics of expression of activation markers on CD4+ Cells in healthy control after stimulation with PHA mitogen (n=25)|
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|Figure 4: Kinetics of expression of activation markers on CD8+ Cells in healthy control after stimulation with PHA mitogen (n=25)|
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|Table 1: The Mean ± SD percentage values of both (CD4+) and (CD8+) cells expressing activation markers before and after stimulation with PHA mitogen|
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The baseline expression of activation markers on CD4 + cells and CD8 + cells before and after stimulation with Brucella antigen in normal subject showed a very low level or no expression of any of the activation markers tested compared to the stimulated samples with PHA (P < 0.001) as shown in [Table 2]. In the patients with active brucellosis, however, in vitro stimulation of T-lymphocyte using Brucella antigen resulted in significant upregulation of the markers and the percentage of both T-cells expressing these activation antigens [Table 3] and [Figure 5] and [Figure 6]. The percentage of CD4 positive cells expressing the different markers in ranking frequencies as depicted in [Figure 5] were as follows: CD38 (mean 31.5%), CD25 (mean 28.7%), CD69 (27.5%), and CD71 (22.7%). The percentage of cells CD8 positive cells expressing activation markers in decreasing order of expression in [Figure 6] were: CD38 (mean 33.6%), then CD69, CD25, and CD71 showed values of mean 28.9%, 26.1%, and 23.4%, respectively. The un-stimulated sample did not show any expression of any of the activation markers tested (P < 0.001).
|Figure 5: Activation of peripheral blood mononuclear cells with expression of activation markers on CD4+ cells from patients with Brucellosis before and after stimulation with Brucella antigen (n=15)|
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|Figure 6: Activation of peripheral blood mononuclear cells with expression of activation markers on CD8+ cells from patients with Brucellosis before and after stimulation with Brucella antigen (n=15)|
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|Table 2: The mean percentage values of expressing T-Cell activation markers on CD4+ and CD8+ of normal donors before and after stimulation with Brucella antigen over 4 days|
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|Table 3: The mean percentage values of different activation marker expressed on CD4 + and CD8+ cells of Brucella patients before and after stimulated with Brucella antigen|
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| Discussion|| |
As an alternative to the lymphocyte transformation test, this study proposes evaluation of the expression of activation markers in response to Brucella antigen as a diagnostic test for seronegative Brucellosis. Antigen induced lymphoproliferative assay gives information only about the overall proliferative responses without detailing the specific cell subset involved in these responses. Furthermore, the procedure is time consuming, requires sophisticated equipment, and technical expertise. Hence, it will be difficult to adopt it as a routine diagnostic test. Expression of activation molecules prior to proliferation, on the other hand, offers a useful method to predict lymphocyte proliferative activity. This is so as the upregulation of these molecules had been shown to correlate well with the cell proliferation.  CD69 expression had been evaluated in several studies with consistent result as a reliable predictor of proliferation of T cells in response to a variety of stimuli. ,,
Brucella antigen induced upregulation of CD69 on T-lymphocytes as an early marker of activation had been well documented in patients with active Brucellosis with a serological evidence of Brucella infection. , This study, when compared to a previously published case report,  is the first ever assessment of expression of activation markers including CD69 in a relatively large number of patients with active Brucellosis without any serological evidence of the infection. This study had demonstrated that antigen induced upregulation of activation markers in seronegative patients with brucellosis may serve as a useful marker for diagnosis of the infection particularly in this subset of patients.
CD38 is a type II transmembrane glycoprotein and plays a role in lymphocyte adhesion, proliferation, and cytokine production.  Most peripheral blood, T and B-cells, as well as red blood cells, are CD38 negative. Along with CD69, CD38 is not expressed on resting lymphoid cell population. , However, CD38 and CD69 are rapidly induced on the surface of lymphocytes following activation and both the molecules have been regarded as a marker for activation of T-cell. , In this study, CD69 expression was not detected in 25 subjects at 0 hours following stimulation with PHA mitogen. However, after 24 hours of culture with a mitogen, an increase in CD69 expression was detectable (25% and 23%) on CD4 and CD8 cells, respectively; peaking at 48 h and 72 h. Previous studies reported different findings with the changes in the percent of T-cells expressing CD69 which showed measurable changes after 4 h, peaking at 8 h and declining at 24 h after stimulation with PHA mitogen. , This study had demonstrated that evaluation of CD69 expression at 72 h provide a more sensitive measure of lymphocyte response to a mitogen stimulation than looking for increase in the percentage of CD69 positive cells. On the other hand, CD25 expression was detected at a low level after 24 h of stimulation (8.1%). The difference between the stimulated and un-stimulated cells was statistically significant (P < 0.001). At 96 h, the CD25 marker peaked to (21% and 23%) on both CD4 + and CD8 + cells, respectively. The CD71 marker was also detected in low level after 24 h (11%) and (13%) and peaked at 72 h on CD4 and at 96 on CD8 cells. These findings were comparable with report by Caruso A et al. who showed that the expression of CD25 and CD71 markers peaked between four and eight days after PHA stimulation.  This expression of activation markers on cells appeared at different times which reflect the different stages of activation of lymphocytes.
After the initial evaluation with phytohemagglutinin (PHA), expression of activation markers was then assessed after stimulation with Brucella antigen in these seronegative samples from patients with active brucellosis. Brucella antigen does not primarily have mitogenic activity against normal lymphocytes, but only provokes activation of cells from sensitized individuals infected by the bacteria.  This was also evident from the observation among the control groups where PHA induced a significant upregulation of activation markers, whereas Brucella antigen failed to do so indicating that pre-sensitization with Brucella antigen in seronegative patients is a prerequisite for this test to yield a positive result [Table 2]. Seventy-two hours (72 h) was selected as the optimal time for culture and stimulation with antigen based on the findings in this study with mitogen stimulation which is in agreement with a previous study.  The percentage of expression of various markers was significantly positive in both CD4 + and CD8 + cells in all these seronegative samples, which confirms the role of T- cells in Brucella infection. ,,
| Conclusion|| |
In conclusion, this study had clearly demonstrated that the multi-parameter flow cytometric assay represent a powerful tool to assess cellular response against Brucella organism. It had also re-emphasized the role of T-cells in brucellosis. It had also been shown that measurement of activation markers namely CD69 and CD38 by lymphocyte activation test (LAT) as a very sensitive as well as specific test in the diagnosis of human brucellosis with compatible medical history but negative serology.
| Acknowledgments|| |
This work was supported fully by a grant from King Abdulaziz City for Science and Technology (Government), Riyadh, Saudi Arabia, and to Dr. Abdullah A Abba for his constructive comments on the manuscript.
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Fahad M Almajid
Department of Medicine, King Khalid University Hospital, P.O.Box: 2959, Riyadh 11461
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]