Clinical and laboratory features of brucellosis in a university hospital in Tunisia.

AIM OF THE STUDY: The increase in the number of brucellosis cases between 2014 and 2017 (14 and 90 cases respectively) led us to study the biological and clinical-epidemiologic characteristics patients hospitalized in Rabta hospital of Tunis. MATERIAL AND METHODS: This retrospective study was conducted in Rabta Hospital in Tunis between 2016 and 2017. It includes 131 patients who had a positive bacteriological diagnosis of Brucella between 2016 and 2017. Diagnosis of brucellosis was made in blood culture by using Bactalert (Biomerieux®). Identification of Brucella was realized by Gram staining, catalase, oxydase. Serological diagnosis was made by testing sera for brucellosis agglutinins with Rose Bengale and the standard agglutination test. The collected data were analyzed by SPSS softcare version 24. RESULTS: The prevalence of Brucellosis in Rabta hospital increases from 14 cases in 2014 to 90 cases in 2017. The mean age was 45 years and ages range from 16 to 84 years. Rural origin was found in 75 cases (68%). Ninety-seven patients (89%) were hospitalized in the infectious diseases department. The average length of hospitalization was 17.25 days. Fifty-seven patients (52%) had a history of consuming unpasteurized dairy products and 45 (41%) were farmers. Fever was the predominant symptom in 104 cases (95%). Osteoarticular involvement is the most common complication of brucellosis and it occurred in 28% of patients. Blood cultures were 73 cases and 42 (57%) were positive for Brucella spp. Rose Bengale was positive in 100% of cases. High titles of the standard agglutination test (superior to 1/1280) were noted in 24 cases (22%). CONCLUSION: Brucellosis is still endemic In Tunisia. Contact with domestic animals and consumption of raw milk and milk products seems to be the major mode of transmission. Control of animal infection by vaccination, occupational and personal hygiene, farm sanitation and preventive measures can reduce disease incidence.

Disseminated histoplasmosis diagnosed in the bone marrow of an HIV-infected patient: First case imported in Tunisia.

Histoplasmosis is a fungal infection caused by a dimorphic fungus, Histoplasma capsulatum. We report a first case of disseminated histoplasmosis in a 34-year-old woman, infected with human immunodeficiency virus (HIV), originating from Ivory Coast and living in Tunisia for 4 years. She was complaining from fever, chronic diarrhoea and pancytopenia. The Histoplasma capsulatum var. capsulatum was identified by direct microscopic examination of the bone marrow. She was treated by Amphotericin B, relayed by itraconazole. Even though a regression of symptoms and normalization of blood cell count (BCC), the patient died in a respiratory distress related to CMV hypoxemic pneumonia.

Asymptomatic intestinal carriage of microsporidia in HIV-positive patients in Tunisia: prevalence, species, and pathogenesis.

Asymptomatic carriage of microsporidia (ACM) has not been described in patients living with HIV (PLHIV) in Tunisia. To determine the prevalence of ACM in PLHIV followed at Tunis la Rabta hospital, describe its clinical features and course, and identify the species involved. This prospective study (2005-2009) included 71 asymptomatic PLHIV compared with 37 PLHIV with diarrhea. One stool sample per patient was examined by microscopy after Weber staining and by PCR. Species identification was confirmed by specific PCR and sequencing. In cases of ACM, a second stool sample was examined in 2010 and a clinical check-up took place in 2013. The prevalence of ACM in asymptomatic PLHIV was 11.3 % (8/71). PCR was more sensitive than microscopy (P = 0.0047). ACM was associated with stage C of HIV infection (P = 0.008) and CD4 T cells <100/µl (P = 0.033). The species involved were E. intestinalis (6 cases) and E. bieneusi (2 cases). Six PLHIV remained asymptomatic with negative stool examinations, but two developed digestive signs. ACM is common among Tunisian PLHIV and it appears to be associated with E. intestinalis.

Isolation of Mycoplasma meleagridis from chickens.

Mycoplasma meleagridis (MM) is a major cause of disease and economic loss in turkeys. Formerly it was thought that this species was very host specific and only restricted to turkey. In this study, we report on the recovery of MM from breeding flocks of chickens located near a turkey breeding unit. Ten MM field strains were isolated (by culture on Frey broth medium) from tracheal swabs of chickens displaying clinical signs of mycoplasmosis-essentially respiratory symptoms and poor performance. Assignment of the isolated field strains to MM was confirmed by a growth inhibition assay using MM-specific polyclonal antiserum and by PCR amplification targeting the 16S rRNA sequence as well as the Mm14 sequence, a MM-species-specific DNA fragment previously identified and characterized in our laboratory. The nucleotide sequence of Mm14 proved to be highly conserved among the 10 MM field strains, indicating a common source of infection. However, on the basis of slight differences in sodium dodecyl sulfate-polyacrylamide gel electrophoresis whole-cell proteins and western blot profiles, two groups of the isolated MM field strains could be distinguished. Evidence of MM infection of chickens was further provided by serology, since 13.77% (35/254) of sera proved positive to MM by either rapid serum agglutination or recombinant antigen-based enzyme-linked immunosorbent assay. In addition, sera of all chickens from which MM was isolated were positive for antibodies to MM. Collectively, the data unambiguously show that MM could infect chickens; thus, MM warrants further exploration to determine its pathogenicity in this unusual host.

Radioimmunolocalisation of bladder tumors xenotransplanted in nude mice.

We have previously reported on the derivation of mouse monoclonal antibodies (Mabs), identifying several cell surface antigens selectively associated with cancer of the urinary bladder (TCC) (1-4). Three of these Mabs (4E8, SK4H and 8F4) have now been assessed for their ability to localise TCC-tumor xenografts in nude mice. The biodistribution of 125I-labeled intact Mabs as well as the corresponding Fab and F(ab')2 fragments from two of them were investigated in animals carrying TCC tumors or antigen negative control tumors. Using direct measurements of excised tissues, all three antibodies were seen to accumulate specifically in the TCC tumors, giving tumor to normal tissue ratios of between 3 and 20 depending on the Mab used and the time after injection. Antibody fragments were generally more efficient in their localisation, mainly due to a dramatic reduction in the blood background as compared to intact Ig. One of the antibodies, 4E8, was also employed for external imaging with gamma camera scintigraphy using 111In or 131I as tracers. Excellent visualisation of the tumor sites could be obtained both with Fab fragments and intact antibody within 12-24 hours after injection. As expected, background radioactivity was significantly lower with fragments than with whole molecules. 111In labeled antibodies appeared in all instances to be superior to the corresponding 131I conjugates. In conclusion, the present study indicates that the three anti TCC antibodies may become useful for the in vivo diagnosis of bladder cancer in man.

Human bladder cancer associated antigens: evaluation of antigenicity in TCC tissues of different grades and in normal urothelium.

The expression of five antigens, associated with transitional cell carcinoma (TCC) of the urinary bladder on biopsies of tumors or normal urothelium, was studied by immunostaining with the corresponding monoclonal antibodies. Both tissue sections and single cell preparations were investigated with either indirect immunoperoxidase staining or immunofluorescence. All 5 antigens were expressed on the majority (70-90%) of sectioned tumor specimens from 44 TCC patients, and 4 of them were similarly expressed on single cell tumor preparations from 26 additional patients. However, in both types of preparation, the degree of expression of these antigens varied from scattered staining of less than 25% of the tumor cells to homogenous staining of all or almost all cells. This degree of expression varied individually for each of the antigens and was not related to the malignancy grade of the tumors. However, as most of the tumors were of grades II or III, no conclusions regarding the relationship of antigen expression to the aggressiveness of the tumors can be drawn. In any event, all tumors expressed at least one and mostly several of these antigens. Antigen expression on biopsies of normal bladder mucosa from TCC patients or on urothelial biopsies from patients with prostate hyperplasia was also observed on single cell specimens (34 patients) but not on sectioned material (9 patients). However, the frequency of positive specimens was much lower (4-20%). Moreover, the number of cells expressing one or, occasionally, several of the antigens in normal urothelium was small (usually less than 5%). Because of these marked differences in antigen expression between tumors and normal tissue, the results indicate that a combination of 3-5 of the antibodies used in this study may be suitable for diagnostic purposes.

Monoclonal antibodies against carcinoma cells of the human urinary bladder: immunohistochemical staining of tissues.

We have previously described the generation of mouse monoclonal antibodies (Mabs) directed to cell surface antigens of human bladder carcinoma. Based on experiments with cultured cells and a limited number of freshly isolated tissues, four distinct antigens were identified as being associated with this disease. In the present investigation, comprising the immunostaining of tissues of normal, malignant, and fetal origin, we have confirmed and extended the close association of these antigens with bladder cancer. Antibodies to all four antigens could clearly discriminate between malignant and normal uroepithelium. Two of the antibodies, SK4H-12 and 4E8, showed no additional reaction when tested with various adult tissues of normal or malignant origin. Antibodies to the remaining two antigens gave a positive staining of a few other tissue types.

Specificities and binding properties of 2 monoclonal antibodies against carcinoma cells of the human urinary bladder.

Mice were immunized with cultured cells derived from transitional cell carcinoma of the human urinary bladder (TCC). Spleen cells were fused with mouse myeloma cell line Sp2/0-Ag14 and the hybridomas obtained screened for antibody production against a panel of human cells. Two hybridomas were selected for further studies. The antibodies from one of these hybridomas (P7A5-4) could clearly discriminate between malignant and normal cells from the bladder, both when tested with cultured cells and fresh tissue. The P7A5-4 antibodies, however, also reacted with some non-TCC cultured carcinoma and melanoma cells but to a lesser extent. This difference in reactivity was even more pronounced in the fresh tumours tested, thus indicating a quantitative difference in antigen expression between TCC and other cells. From extracts of TCC cells, P7A5-4 bound three polypeptides of mol. wts 92Kd (ConA+), 23 and 17Kd (ConA-). The antibody derived from hybridoma SK4H-12 bound a ConA reactive glycopeptide of 100Kd mol. wt, the expression of which was almost entirely restricted to urothelial cell lines and tissue of TCC origin, as shown by immunocytochemical studies. The finding in this study of new antigens associated with urinary bladder carcinoma, extend the results obtained previously in our laboratory (Koho et al., 1984; Paulie et al., 1984) and further delineate the heterogeneity of tumour-associated antigens in this human tumour system.

A p50 surface antigen restricted to human urinary bladder carcinomas and B lymphocytes.

We have previously described the derivation of a monoclonal antibody, S2C6, to a novel 50 Kdalton antigen associated with human urinary bladder carcinoma. No reactions were obtained with carcinomas of unrelated origin or with normal urothelial cells. However, the antibody also reacted with a similar antigen on some cell lines of B lymphocyte origin. Using large panels of target cells we have now shown that this reactivity was entirely restricted to cells of the B lineage within the haematopoietic system. As opposed to its apparent restriction to malignant cells of the urothelium, the S2C6 antigen was expressed by normal B lymphocytes as well as by many malignant B cells (chronic lymphocytic leukaemia, hairy cell leukaemia and immunocytoma). Pre-B cells derived from acute lymphocytic leukaemia and plasma cells from multiple myeloma lacked the antigen. Expression was significantly enhanced on cultured B cells from Burkitt lymphomas and on Epstein-Barr virus-transformed lymphoblastoid cell lines including those of the pre-B phenotype derived from fetal bone marrow. As judged from the molecular size and the distribution pattern displayed by the S2C6 antigen it appears to be distinct from other B cell antigens previously described. A possible relation of the S2C6 antigen to a receptor for B cell growth factors is discussed.

Production of antibodies to cellular antigens by EBV-transformed lymphocytes from patients with urinary bladder carcinoma.

Lymphocytes from patients with transitional-cell carcinoma (TCC) of the urinary bladder were transformed by infection with Epstein-Barr virus. To obtain B cells secreting antibodies reactive with TCC cells, the transformed cells were either adhered to irradiated monolayers of cultured allogeneic TCC cells or subcultured at limiting dilution. Supernatants from these cultures were tested in a modified enzyme-linked immunosorbent assay against fixed cells, isolated plasma membranes, or lipid antigens or were tested by antibody-dependent cellular cytotoxicity (ADCC). Reactions with antigens derived from the serum source were excluded by proper controls. By this approach a majority of the patients tested (7/12) gave rise to cultures producing antibodies recognizing various cellular antigens. The antibody-containing supernatants from these cultures were usually of high titres and the reactive antibodies of IgM isotype. One culture, which had been selected by repeated adherence to TCC cells, produced antibodies reactive with such cells in ADCC. None of the antibodies investigated detected antigens exclusively present on TCC cells.

Monoclonal antibodies to antigens associated with transitional cell carcinoma of the human urinary bladder. I. Determination of the selectivity of six antibodies by cell ELISA and immunofluorescence.

Spleen cells from BALB/c mice immunized with cells derived from transitional cell carcinomas (TCC) of the human urinary bladder were fused with mouse myeloma Sp 2/0 Ag14 cells. Monoclonal antibodies from six established hybridomas were investigated for specificity in a cell ELISA and in indirect immunofluorescence against a large panel of fixed intact cells. Three of the antibodies reacted with half or more of the eight bladder tumors and with a few unrelated tumors. They did not react at all with malignant or normal cells of hematopoietic origin. A fourth antibody reacted with seven of eight bladder tumors. It also reacted weakly with a prostatic carcinoma, with five of six malignant or transformed B cell lines, and with a subpopulation of normal lymphocytes, but not with any of the other cells on the test panel. These four antibodies did not react with cells derived from normal urothelium. The results suggest that these antibodies might recognize cell-type-restricted antigens associated with malignancy. Another antibody reacted with almost all urothelium-derived cells. It also reacted with three of three melanomas but not with any other cells on the panel. The sixth antibody reacted with 32 of the 37 cells tested. The spectrum of reactivities displayed by the antibody suggested that it recognizes HLA antigens.

Monoclonal antibodies to antigens associated with transitional cell carcinoma of the human urinary bladder. II. Identification of the cellular target structures by immunoprecipitation and SDS-PAGE analysis.

The cellular target structures for six monoclonal antibodies raised against cultured human bladder carcinoma cells (TCC) were investigated. The specificities of these antibodies when tested against a large panel of cells have been described in the companion paper. Radiolabeled cell lysates were precipitated with the different monoclonal antibodies bound to protein A (Staphylococcus aureus) on a matrix of Sepharose beads. The precipitates were separated by sodium dodecyl sulfate- gel electrophoresis (SDS-PAGE) and analyzed by autoradiography. The antibodies 4B5, 7E9, and 14B11 have previously been found to react in a similar way with TCC-targets and some non-TCC tumor cells, but not with normal urothelial cells or cells of hematopoietic origin. When tested with lysates of a TCC-cell line (TCCSuP) a strong 92K band and a weak 23K band were precipitated with any one of these antibodies. These polypeptides were expressed on the cell surface and were not linked by disulfide bonds. Depletion experiments confirmed that the three antibodies recognized the same antigens. Another antibody (4E8) probably directed to a differentiation antigen present on both urothelial and melanoma cells detected two high molecular polypeptides, 190K and 170K. Antibodies from the S2C6 hybridoma, which displayed a distinct dual specificity for TCC- targets and for malignant or transformed cells of B cell origin, precipitated a 50K component from extracts of either TCC- or B cell-derived cell lines. Antibodies produced by the S2A9 hybridoma were shown to bind to a framework epitope of the HLA-A, B, C heavy chain.