Development and validation of a novel diagnostic test for human brucellosis using a glyco-engineered antigen coupled to magnetic beads.

Brucellosis is a highly contagious zoonosis and still a major human health problem in endemic areas of the world. Although several diagnostic tools are available, most of them are difficult to implement especially in developing countries where complex health facilities are limited. Taking advantage of the identical structure and composition of the Brucella spp. and Yersinia enterocolitica O:9 O-polysaccharide, we explored the application of a recombinant Y. enterocolitica O:9-polysaccharide-protein conjugate (OAg-AcrA) as a novel antigen for diagnosis of human brucellosis. We have developed and validated an indirect immunoassay using OAg-AcrA coupled to magnetic beads. OAg-AcrA was produced and purified with high yields in Y. enterocolitica O:9 cells co-expressing the oligosaccharyltransferase PglB and the protein acceptor AcrA of Campylobacter jejuni without the need for culturing Brucella. Expression of PglB and AcrA in Y. enterocolitica resulted in the transfer of the host O-polysaccharide from its lipid carrier to AcrA. To validate the assay and determine the cutoff values, a receiver-operating characteristic analysis was performed using a panel of characterized serum samples obtained from healthy individuals and patients of different clinical groups. Our results indicate that, using this assay, it is possible to detect infection caused by the three main human brucellosis agents (B. abortus, B. melitensis and B. suis) and select different cutoff points to adjust sensitivity and specificity levels as needed. A cutoff value of 13.20% gave a sensitivity of 100% and a specificity of 98.57%, and a cutoff value of 16.15% resulted in a test sensitivity and specificity of 93.48% and 100%, respectively. The high diagnostic accuracy, low cost, reduced assay time and simplicity of this new glycoconjugate-magnetic beads assay makes it an attractive diagnostic tool for using not only in clinics and brucellosis reference laboratories but also in locations with limited laboratory infrastructure and/or minimally trained community health workers.

Dynamics of adrenal steroids are related to variations in Th1 and Treg populations during Mycobacterium tuberculosis infection in HIV positive persons.

Tuberculosis (TB) remains the most frequent cause of illness and death from an infectious agent, and its interaction with HIV has devastating effects. We determined plasma levels of dehydroepiandrosterone (DHEA), its circulating form DHEA-suphate (DHEA-s) and cortisol in different stages of M. tuberculosis infection, and explored their role on the Th1 and Treg populations during different scenarios of HIV-TB coinfection, including the immune reconstitution inflammatory syndrome (IRIS), a condition related to antiretroviral treatment. DHEA levels were diminished in HIV-TB and HIV-TB IRIS patients compared to healthy donors (HD), HIV+ individuals and HIV+ individuals with latent TB (HIV-LTB), whereas dehydroepiandrosterone sulfate (DHEA-s) levels were markedly diminished in HIV-TB IRIS individuals. HIV-TB and IRIS patients presented a cortisol/DHEA ratio significantly higher than HIV+, HIV-LTB and HD individuals. A positive correlation was observed between DHEA-s and CD4 count among HIV-TB individuals. Conversely, cortisol plasma level inversely correlated with CD4 count within HIV-TB individuals. M. tuberculosis-specific Th1 lymphocyte count was increased after culturing PBMC from HIV-TB individuals in presence of DHEA. We observed an inverse correlation between DHEA-s plasma level and Treg frequency in co-infected individuals, and CD4+FoxP3+ Treg frequency was increased in HIV-TB and IRIS patients compared to other groups. Strikingly, we observed a prominent CD4+CD25-FoxP3+ population across HIV-TB and HIV-TB IRIS patients, which frequency correlated with DHEA plasma level. Finally, DHEA treatment negatively regulated FoxP3 expression without altering Treg frequency in co-infected patients. These data suggest an enhancing role for DHEA in the immune response against M. tuberculosis during HIV-TB coinfection and IRIS.

Prepatellar bursitis due to Brucella abortus: case report and analysis of the local immune response.

A case of prepatellar bursitis in a man with chronic brucellosis is presented. Brucella abortus biotype 1 was isolated from the abundant yellowish fluid obtained from the bursa. Clinical and epidemiological data did not suggest a direct inoculation of the agent in the bursa. However, the patient mentioned occasional local trauma due to recreational sports, which may have constituted a predisposing factor. As determined by ELISA, there were higher levels of IgG against Brucella LPS and cytosolic proteins detected in the patient's bursal synovial fluid when compared with serum. Levels of proinflammatory cytokines (tumour necrosis factor alpha, interleukin 1 beta, gamma interferon, interleukin 8 and MCP-1) were higher than in synovial fluids obtained from patients with rheumatoid arthritis and a patient with septic arthritis, and a zymographic analysis revealed a gelatinase of about 92 kDa. These findings indicate that it may be possible to diagnose brucellar bursitis by measuring specific antibodies in the bursal synovial fluid. In addition, our findings suggest a role of increased local levels of proinflammatory cytokines and gelatinases in the inflammatory manifestations of brucellar bursitis.

Brucella abortus inhibits major histocompatibility complex class II expression and antigen processing through interleukin-6 secretion via Toll-like receptor 2.

The strategies that allow Brucella abortus to survive inside macrophages for prolonged periods and to avoid the immunological surveillance of major histocompatibility complex class II (MHC-II)-restricted gamma interferon (IFN-gamma)-producing CD4+ T lymphocytes are poorly understood. We report here that infection of THP-1 cells with B. abortus inhibited expression of MHC-II molecules and antigen (Ag) processing. Heat-killed B. abortus (HKBA) also induced both these phenomena, indicating the independence of bacterial viability and involvement of a structural component of the bacterium. Accordingly, outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, inhibited both MHC-II expression and Ag processing to the same extent as HKBA. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited MHC-II expression, indicating that any Brucella lipoprotein could down-modulate MHC-II expression and Ag processing. Inhibition of MHC-II expression and Ag processing by either HKBA or lipidated Omp19 (L-Omp19) depended on Toll-like receptor 2 and was mediated by interleukin-6. HKBA or L-Omp19 also inhibited MHC-II expression and Ag processing of human monocytes. In addition, exposure to the synthetic lipohexapeptide inhibited Ag-specific T-cell proliferation and IFN-gamma production of peripheral blood mononuclear cells from Brucella-infected patients. Together, these results indicate that there is a mechanism by which B. abortus may prevent recognition by T cells to evade host immunity and establish a chronic infection.

Human infection with M- strain of Brucella canis.

The less mucoid strain of Brucella canis or M- strain is used for the serologic diagnosis of canine brucellosis. While this strain is avirulent in dogs, we report the case of clinical brucellosis that developed in a laboratory worker a few days after handling live M- cells for antigen production.

Antibody reactivity to Omp31 from Brucella melitensis in human and animal infections by smooth and rough Brucellae.

Group 3 of outer membrane proteins (OMPs) of Brucella includes Omp25 and Omp31, which share 34% identity. Omp25 is highly conserved in Brucella species, and Omp31 is present in all Brucella species, except Brucella abortus. Antibodies to Brucella melitensis Omp31 have been sought only in infected sheep, and Western blotting of sera from infected sheep did not reveal anti-Omp31 reactivity. We obtained recombinant purified Omp31 (B. melitensis) and tested its recognition by sera from humans and animals suffering from brucellosis by an indirect enzyme-linked immunosorbent assay (ELISA). Serum samples from 74 patients, 57 sheep, and 47 dogs were analyzed; brucellosis was confirmed by bacteriological isolation in all ovine and canine cases and 31 human cases of brucellosis. Thirty-five patients (47%) were positive for antibodies to Omp31, including seven cases of Brucella suis infection, two cases of B. abortus infection, and three cases of B. melitensis infection. Of 39 sheep naturally infected with B. melitensis (biovars 1 and 3), 23 (59%) were positive for antibodies to Omp31. Anti-Omp31 antibodies were also detected in 12 of 18 rams (67%) in which Brucella ovis was isolated from semen. Antibodies to Omp31 were also found in 41 (87%) of the 47 dogs, including 13 with recent infection. These results suggest that an indirect ELISA using recombinant purified Omp31 from B. melitensis would be of limited value for the diagnosis of human and animal brucellosis. Nevertheless, the potential usefulness of this antigen in combination with other recombinant proteins from Brucella should not be dismissed.

Diminished production of T helper 1 cytokines correlates with T cell unresponsiveness to Brucella cytoplasmic proteins in chronic human brucellosis.

This study evaluated the cellular immune response against Brucella species cytoplasmic protein (CP) in peripheral blood mononuclear cells (PBMC) of 25 patients with brucellosis. In vitro proliferation and cytokine gene expression and production were investigated. PBMC from 14 patients proliferated in response to CP (responder patients [RPs]) and cells from 11 patients did not (nonresponder patients [NRPs]). CP-specific interleukin (IL)-2 and interferon-gamma were significantly induced in PBMC from RPs, compared with cells from NRPs. No significant differences were found in the production of IL-10 between the 2 groups. CP did not induce IL-4 production. A close relationship was observed between the clinical status of the patients and the T cell response against CP. Patient with acute infections responded to CP and induced production of T helper 1 (Th1) cytokines, whereas chronically infected patients did not. Diminished production of Th1 cytokines may contribute to T cell unresponsiveness in chronic human brucellosis.

Serodiagnóstico de la brucelosis humana por aglutinación directa: problemas de interpretación causados por discrepancias entre resultados obtenidos con diferentes antígenos comerciales / Serodiagnosis of human brucellosis using agglutination tests: misinterpretation caused by discrepancies among results obtained with different commercial antigens

En este trabajo se comparó mediante ensayos de aglutinación, la reactividad de un mismo grupo de sueros frente a las distintas suspensiones comerciales de Brucella. Se determinó el volumen celular de 5 antígenos comerciales para aglutinación en placa (Huddleson), 1 antígeno comercial para reacción de Rosa de Bengala y los antígenos producidos por el Instituto Panamericano de Protección de Alimentos y Zoonosis (INPPAZ). Se observaron diferencias importantes de volumen celular entre los antígenos para Huddleson, pero no entre los antígenos para Rosa de Bengala. Con la reacción de Huddleson se ensayaron 11 sueros reactivos y 25 sueros de individuos no infectados. Se obtuvieron títulos aglutinantes más elevados con los antígenos de menor volumen celular que con los de mayor volumen celular. Resultados similares se obtuvieron al ensayar por aglutinación en tubo 8 sueros reactivos. Cuando se analizaron por Rosa de Bengala 24 sueros reactivos y 25 controles, los resultados correlacionaron en un 100 por ciento. Este estudio demuestra que existen diferencias de concentración celular importantes entre los antígenos analizados, confirma que dichas diferencias generan variaciones en el título aglutinante anti-Brucella obtenido con cada suero, y muestra cómo esas variaciones pueden dificultar el establecimiento de un correcto diagnóstico de la enfermedad

Serodiagnóstico de la brucelosis humana por aglutinación directa: problemas de interpretación causados por discrepancias entre resultados obtenidos con diferentes antígenos comerciales / Serodiagnosis of human brucellosis using agglutination tests: misinterpretation caused by discrepancies among results obtained with different commercial antigens

En este trabajo se comparó mediante ensayos de aglutinación, la reactividad de un mismo grupo de sueros frente a las distintas suspensiones comerciales de Brucella. Se determinó el volumen celular de 5 antígenos comerciales para aglutinación en placa (Huddleson), 1 antígeno comercial para reacción de Rosa de Bengala y los antígenos producidos por el Instituto Panamericano de Protección de Alimentos y Zoonosis (INPPAZ). Se observaron diferencias importantes de volumen celular entre los antígenos para Huddleson, pero no entre los antígenos para Rosa de Bengala. Con la reacción de Huddleson se ensayaron 11 sueros reactivos y 25 sueros de individuos no infectados. Se obtuvieron títulos aglutinantes más elevados con los antígenos de menor volumen celular que con los de mayor volumen celular. Resultados similares se obtuvieron al ensayar por aglutinación en tubo 8 sueros reactivos. Cuando se analizaron por Rosa de Bengala 24 sueros reactivos y 25 controles, los resultados correlacionaron en un 100 por ciento. Este estudio demuestra que existen diferencias de concentración celular importantes entre los antígenos analizados, confirma que dichas diferencias generan variaciones en el título aglutinante anti-Brucella obtenido con cada suero, y muestra cómo esas variaciones pueden dificultar el establecimiento de un correcto diagnóstico de la enfermedad (AU)