Zouheir Bahloul

Clinical and diagnostic value of ribosomal P autoantibodies in systemic lupus erythematosus

OBJECTIVE To analyse prospectively the diagnostic sensitivity and specificity as well as the clinical relevance of ribosomal P (anti-P) autoantibodies in a large cohort of SLE patients. METHODS The anti-P autoantibodies were evaluated in the serum of 200 Tunisian SLE patients at disease onset and 130 various control subjects by a sensitive immunodot assay. A complete laboratory evaluation and clinical examination were performed in each SLE patient. During the follow-up, the patients were regularly monitored for clinical parameters. Global SLE activity was measured by the ECLAM. RESULTS The sensitivity and specificity of anti-P testing for SLE were 23.5 and 98.4%, respectively. The anti-P-positive samples 14/47 (29.8%), 27/47 (57.4%) and 5/47 (10.6%) were negative for anti-dsDNA, anti-Sm or both antibodies, respectively. The anti-P-positive patients showed more active disease activity and a much higher prevalence of arthritis. An association between IgG aCLs and anti-P antibodies was also found. However, anti-P antibodies were not associated with neuropsychiatric manifestations or lupus nephritis. CONCLUSION This study does not seem to confirm the described association of anti-P antibodies with neuropsychiatric manifestations of SLE. However, it supports the anti-P antibody association with arthritis and disease activity as well as the presence of aCL. Based on our study and other related studies, we propose that, akin to anti-Sm and anti-dsDNA, anti-P antibodies detected by one agreed method may be considered for inclusion as a criterion for the classification of SLE.

Association study of human leukocyte antigen-DRB1 alleles with rheumatoid arthritis in south Tunisian patients

The aim of this study is to explore relationship between HLA-DRB1 alleles and the susceptibility and clinical features of rheumatoid arthritis (RA) in the south Tunisian population. We studied 142 RA patients and 123 controls matched for age, sex, and ethnicity. HLA-DRB1 genotyping and HLA-DRB1*04 subtypes were performed using polymerase chain reaction/sequence-specific primers. Association was assessed based on the χ2 test and odds ratio with 95% confidence interval. For multiple comparisons, p value was corrected (pc) with Bonferroni test. Two alleles, HLA-DRB1*04 (p = 0.045, pc = NS) and HLA-DRB1*10 (p = 0.021, pc = NS), were found to have increased frequencies in RA patients compared to controls. In contrast HLA-DRB1*08 allele was found to have a decreased frequency in patients compared to controls (p = 0.044, pc = NS). Molecular subtyping of the most prevalent allele (DRB1*04) revealed increased frequencies of HLA-DRB1*04:05 in patients compared to controls (p = 0.013, pc = NS) whereas HLA-DRB1*04:02 showed a protective effect (p = 0.005, pc = 0.04). Moreover, stratified analyses indicated statistically significant associations between HLA-DRB1*04 allele and anti-cyclic peptides antibodies positivity (ACPA+) and rheumatoid factor positivity (RF+; pc = 0.03, for both subgroups), HLA-DRBI*10 and ACPA+ and the presence of another autoimmune disease (pc = 0.05 and pc = 0.007, respectively), and HLA-DRB1*04:05 and RF+ and erosion (pc = 0.005 and pc = 0.049; respectively). A significant decrease in the frequency of the DRB1*04:02 allele was observed in patients with ACPA+ and RF+ subgroups (pc = 0.04 and pc = 0.02, respectively). Our results showed that there was a trend of positive association of HLA-DRB1*04 and HLA-DRB1*10 with RA as such and significant associations with the disease severity in the south Tunisian population.

Deep vein thrombosis associated with acute brucellosis: a case report and review of the literature

To the Editor, Human brucellosis remains the most common zoonotic disease worldwide, with more than 500,000 new cases annually. It is still a serious and uncontrolled public health problem in many countries, including Tunisia. Human brucellosis is a multisystem disease that may present with a broad spectrum of clinical manifestations and its complications can affect almost all organs and systems, with varying incidence. Vascular complications of Brucella infection have rarely been reported in the medical literature. By searching MEDLINE, eight cases of deep vein thrombosis (DVT) associated with acute brucellosis have been reported [1-8]. We describe a new case of DVT in the left leg in association with acute brucellosis. A 52-year-old Tunisian man was admitted to our hospital with a 4-day history of fever and left calf pain associated with myalgia and night sweats. He also reported the ingestion of raw sheep milk. The patient was febrile, to 38.3℃. Homan sign was positive on the left. The circumference of his left calf was 3 cm larger than that of the right. Palpation of the right sacroiliac joint was painful. The rest of the physical examination was unremarkable. A Doppler ultrasound study showed a thrombosis in the left femoral vein. Laboratory examinations revealed the following results: white blood cell count 7,300 cells/mm3 (neutrophils, 49%), platelet count 275,000/mm3, erythrocyte sedimentation rate 30 mm, and C-reactive protein 43 mg/dL. Results of other routine biochemical blood tests were all normal. Protein C, protein S, antithrombin III, and activated protein C resistance activity were within normal ranges, and anticardiolipin and lupus anticoagulant were negative. The chest X-ray and abdominal ultrasound were normal. An anteroposterior X-ray of the pelvis showed grade II sacroiliitis of the right sacroiliac joint. Standard tube agglutination testing of initial samples were positive for antibodies to Brucella (titer, 1/640). Based on the history of ingestion raw sheep milk, fever associated with myalgia and night sweats, the presence of sacroiliitis, and a positive Wright test, a diagnosis of acute Brucella infection associated with a DVT in the left leg was made. The patient was treated with rifampicin 900 mg/day and doxycycline 200 mg/day combined with low molecular weight heparin. The treatment durations for both anticoagulant and brucellosis were extended to 3 months. After a follow-up period of 9 months, the patient was asymptomatic and, during this period, no recurrence of brucellosis or DVT was observed. Vascular complications of brucellosis are rare; the arteries are more affected than veins. Arterial complications of brucellosis include aneurysm formation in diverse arteries, such as the aorta, brachial, tibioperoneal, and cerebral arteries, with or without underlying endocarditis. Cutaneous vasculitis has also been noted. Vein thrombosis is a rare vascular complication in acute brucellosis. In 1973, Romem et al. [8] reported the first case of brucellosis complicated with central vein thrombosis. Since this publication, seven further cases of vein thrombosis due to brucellosis have been reported. Table 1 shows demographic data and clinical and laboratory findings of the eight case reports of brucellosis complicated with vein thrombosis. Thrombosis involved the portal vein [7], the central retinal vein [8], the cerebral vein [6], and the lower limb veins [4] in one case each. Acute brucellosis was associated with DVT in the legs in four patients (three men, one woman). The diagnosis of DVT in the leg was made before the onset of other clinical features of brucellosis in one case [2] and concomitant with the diagnosis of brucellosis in two [3,5]. The duration between DVT diagnosis and the occurrence of brucellosis clinical features was 4 weeks. One patient had a history of brucellosis [1]. Table 1 Summary of all reported cases of deep vein thrombosis associated with brucellosis Suggested mechanisms by which brucellosis may cause DVT include induction of inflammation by the infectious process in adjacent tissues, direct endothelial damage, granulomatous endophlebitis, compression from a local soft tissue mass or abscess, induction of a transient hypercoagulable state, and an immune reaction in the vessel wall to a Brucella antigen. In the present case, protein C, protein S, and antithrombin III levels, and activated protein C resistance activity were normal, and no antiphospholipid antibodies were detected. Also, no local infection adjacent to his left leg deep veins was observed in his illness. Thus, it is possible that granulomatous endophlebitis or a possible immune reaction in the vessel wall to a Brucella antigen was responsible for the patients DVT. DVT is a rare complication of brucellosis. We report a new case of leg thrombophlebitis revealing acute brucellosis. The present case and the others reported previously suggest that brucellosis must be considered in patients suffering with DVT, particularly in patients from Brucella-endemic areas.

Association and frequency of HLA-A, B and HLA-DR genes in south Tunisian patients with spondyloarthritis (SpA)

The aim of this study is to investigate the association of HLA-A, B and HLA-DR gene expression and to assess an association of additional HLA antigens besides HLA-B27 in south Tunisian patients with spondyloarthritis (SpA). Eighty-five patients diagnosed with ankylosing spondylitis (AS, n = 68) and reactive arthrithis (ReA, n = 17) were selected and compared with 100 healthy controls (HC). HLA class I antigens were typed serologically using microlymphocytotoxicity technique. HLA-DRB1* alleles were studied by polymerase chain reaction amplification with sequence-specific primers. The significance of differences between patients and controls was tested by chi-square analysis. We found significantly increased frequencies of HLA-A3 (30.6%; pC = 0.04; OR = 2.95), HLA-B27 (62.35%; pC = 4.10−17, OR = 53.55), and HLA-DRB1*15 (17.2%; pC = 0.026; RR = 2.58) alleles in SpA patients compared to HC. The most frequent and strongest association was observed for HLA-B27 in AS (pC = 6.6 × 10−16, OR = 52.23). When AS and ReA patients were analysed separately, HLA-DRB1*15 and HLA-A3 were increased only in AS (pC = 0.01, OR = 2.99 and pC = 0.03, OR = 3.14, respectively). In ReA patients, HLA-DRB1*04 (p = 0.033, pC = NS, OR = 2.89) was found to be the most common allele. By analysing the HLA-B27-negative subgroup, HLA-A3 and HLA-DRB1*15 expression was found to be dependent on the presence of HLA-B27. HLA-B27 expression was higher in male (45/53; 85%) as compared to female (8/53; 15%) patients (p = 0.03). Apart from HLA-B27, HLA-A3 and HLA-DRB1*15 are the MHC class I and II alleles found most frequent in Tunisian patients with AS, whereas HLA-DRB1*04 was found most frequent in ReA patients. HLA-B27 is more frequent in male than in female patients.

Plasma Saturated and Monounsaturated Fatty Acids in Behçet’s Disease

Background: Fatty Acid (FA) composition of serum has been associated with many markers of inflammation. In this study, we tried to examine plasma Saturated Fatty Acid (SFA) and Monounsaturated Fatty Acid (MUFA) composition in Behçets Disease (BD) patients. The associations between the circulating FA levels and some markers of inflammation have also been investigated. Methods: This study is a cross-sectional one. In fact, a total of 101 BD patients and healthy controls group of 99 subjects are enrolled. Gas Chromatograph equipped with a Capillary Split/Splitless Injector and flame ionization detector was used to analyze the plasma SFA and MUFA compositions. The high sensitivity C-Reactive Protein (hsCRP) and fibrinogen levels were measured using standard techniques. Results: BD patients had significantly higher proportions of Mystiric Acid (MA), Palmitic Acid (PAM), Palmitoleic Acid (POA) and Stearoyl-CoA Desaturase (SCD)-16, compared to controls. The results revealed that patients with severe involvements had high levels of POA and total MUFA associated with higher SCD-16 activity compared to those with minor ones. The receiver operator characteristic curve analysis revealed that POA could well discriminate BD patients with severe clinical manifestations. In the bivariate analysis, hsCRP was found to be positively correlated with total SAFA and POA elongase activity index but negatively correlated with SCD-18 activity index. The STA, POA, elongase and SCD-16 activity index are correlated with fibrinogen. On the other hand, the multivariate analysis showed that POA remained associated with higher levels of hsCRP. Conclusion: Unfavourable plasma SFA and MUFA profile were reported in BD patients. POA, which is associated with higher plasma hsCRP level, may play a role in the pathogenesis of BD.

HLA Class III: A susceptibility region to systemic lupus erythematosus in Tunisian population

Background and objectives Short tandem repeats (STR) are usually used as informative polymorphic markers for genetic mapping and for disease susceptibility analysis. The involvement of these microsatellite markers localized in the MHC region was reported in many auto-immune diseases. In this study we analyzed for the first time eight polymorphisms of microsatellite loci at the HLA region: D6S291, D6S273, TNFa, b and c, MICA, D6S265 and D6S276, in Tunisian systemic lupus erythematosus (SLE) patients. Materials and methods We performed a case control study in which the microsatellite loci were amplified using specific primers labeled with NED, VIC, PET or 6-FAM and analyzed using GeneScan software 3.7. For the statistical analysis, we used SPSS software and we performed a sub-haplotype scoring test using the haplo.stats software developed in the R language. Results We found that two mean associated regions existed; the most statistically significant encompassed the 3 TNF markers (p = 0.0003, OR = 19.34); the latter covered the DR region. In fact, when scoring haplotypes in 3 marker- sliding windows, the p value increased as we moved away from the TNF region and decreased again when we approached the DRB1 locus. We also established for the first time the negative association between alleles of D6S291 and SLE. The majority of clinical and serological correlations were noted with TNF alleles. Conclusion Our results confirm the association between TNF and DRB1 polymorphisms and SLE. The association between alleles of D6S291 and SLE needs however to be verified by the analysis of other markers beyond this region.

Measurement of absolute copy number variation of Glutathione S‐Transferase M1 gene by digital droplet PCR and association analysis in Tunisian Rheumatoid Arthritis population

The investigation of copy number variations (CNVs) analysis of candidate genes is currently an important research area in modulating human diseases. We aimed to quantify CNVs in glutathione S‐transferase M1 (GSTM1) gene and determine its genetic contribution in Tunisian rheumatoid arthritis (RA) and its subsets through an innovative technique for quantification.

Beyond Human Leukocyte Antigen Class I Antigens: Hereditary Hemochromatosis Gene Mutations in Recurrent Aphthous Oral Ulcers and Behçet Disease in the South of Tunisia

Objective: The aim of this work was to establish human leukocyte antigen (HLA) class I and hereditary hemochromatosis gene (HFE) mutation associations with recurrent aphthous oral ulcers (RAOU) and Behçet disease (BD) in a cohort of Southern Tunisian patients. Subjects and Methods: A total of 232 patients with RAOU and 123 healthy controls (HCs) were enrolled in this study. The patients were divided into 2 groups based on the presence (BD+: n = 62) or absence of BD (BD−, n = 170). In the BD+ group, 28 patients had severe manifestations of BD. In the BD- group, RAOU was isolated in 81 patients, associated with mucocutaneous manifestations in 58 and with joint symptoms in 25. Complement-dependent microlymphocytotoxicity assay and polymerase chain reaction-restriction fragment length polymorphism were used to study HLA class I polymorphism and HFE mutations, respectively. Results: HLA-B51 was positively associated with BD, particularly in those with severe manifestations. No association was detected with HLA class I polymorphism among the BD group. Based on stratification to clinical manifestations, the isolated RAOU was negatively associated with HLA-A1 with a difference close to significance (12 [14.81%] vs. 32 [26.02%] in HCs; p = 0.06). Furthermore, patients with mucocutaneous features had a higher frequency of HLA-B51 (14, 24.14%) than patients without mucocutaneous involvement (11, 11.37%). Considering HFE mutations, patients with isolated RAOU had a higher frequency of H63D when compared with other subgroups, especially after limiting the comparison to 27 patients of at least 5 years of follow-up. Conclusion: This study showed that, unlike BD, RAOU were not associated with HLA-B51. Moreover, we suggest that H63D mutation was positively associated with isolated RAOU.