Infrequent patterns in cerebrospinal fluid isofocusing test: Clinical significance and contribution of IgG index and Reiber diagram to their interpretation.

Cerebrospinal fluid (CSF) isoelectrofocusing (IEF) is considered as the gold standard for detecting an intrathecal synthesis of IgG, which is a hallmark of multiple sclerosis (MS). This corresponds to the presence of CSF-restricted IgG oligoclonal bands (OCB) (typically type 2 pattern). Moreover, this technique can also detect a systemic immune reaction with passive transfer of IgG (type 3 and 4 patterns) for which the clinical relevance is less understood. The aim of our study was to determine the frequency and disease associations of IEF type 3 and 4 patterns and to investigate the potential usefulness of including quantitative data (IgG index and Reiber Diagram) in interpreting such IEF profiles. Among 544 patients who underwent CSF IEF (Hydragel CSF isofocusing kit, Sebia®, France) in our Laboratory during a six-year-period, those who presented type 3 or 4 patterns were selected. Clinical data and results of other immunological tests were analyzed for 27 patients followed in the Neurological Department. Frequencies of type 3 and type 4 patterns were relatively low (2.3 % and 3.4 % respectively). Among patients with type 3 pattern included in our study (n = 10), 5 were diagnosed with MS. For the 5 other patients, the diagnosis was a clinically isolated syndrome (CIS) (2 cases), a probable auto-immune encephalitis (2 cases) and a possible genetic neurodegenerative disease (1 case). MS patients had an IgG index >0.7 and fell into area 4 of Reiber diagram while non-MS patients had an IgG index <0.7 and fell into area 1, except the last case. Regarding type 4 pattern (n = 17), the diagnoses were as follows: MS (3), CIS (4), Neuromyelitis optica spectrum disorders with positive anti-AQP4 antibodies (3) and anti-NMDAR autoimmune encephalitis (1). The remaining cases had central nervous system impairment related to vascular, metabolic or tumoral etiologies (3) or peripheral nervous system impairment (3). In this group (type 4 pattern), IgG index was <0.7 in 15/17 cases. Interpretation using Reiber diagram showed an abnormal blood-brain barrier for 8/17 patients. Type 3 and 4 IEF patterns are infrequently observed in routine neurology practice. It is important for the diagnostic laboratory professional as well as for the neurologist to understand their clinical relevance. Our findings highlight the contribution of quantitative evaluation of CSF (IgG index, Reiber diagram) for the interpretation of such situations. Despite the small size of our study population, our results emphasize the importance of reporting the exact type of IEF pattern and not only the positivity or not of OCB.

The presence of two light chain bands on immunofixation is associated with poor outcomes in newly diagnosed multiple myeloma patients.

We aimed to describe the clinical and biological characteristics and the prognosis of patients presenting with an additional light chain (LC) band along with a complete monoclonal protein on immunofixation (IF).An 8-year descriptive study was conducted to assess all cases with confirmed monoclonal gammopathies (MG). We studied those with an entire M-protein with 2 bands of LC of the same isotype based on the results of IF. Data were collected from patients' files.Among 548 cases of MG, we found 32 cases (5.8%) with an additional LC band. We included 28 patients (5%) with a confirmed diagnosis of multiple myeloma (MM). The m/f ratio was 2.5 with a median age of 63 years [32-80 years]. All MM patients had anemia, 16 (57%) had renal failure, 14 (50%) had lytic lesions, 9 (32%) received hemodialysis, and 7 (25%) had hypercalcemia. The free-kappa-lambda ratio was abnormal in all cases: median = 0.07 [0.002-58.57]. The mean overall survival (OS) was 22 months ± 38.76.Fifteen MM patients (48%) received chemotherapy, and 7 (22%) autologous stem cell transplants (SCT). Patients who received SCT had an OS higher than those who received other treatments (p = 0.038). OS was low in patients with high ß2microglobulin levels (rho = -0.791; p = 0.001), and abnormally low free-kappa-lambda ratio (rho = -0.852;p = 0.04).The presence of an additional LC band with a complete monoclonal protein seems to identify newly diagnosed MM patients with poor outcomes and frequent renal impairment.

miRNA implication in the pathogenesis and the outcome of Tunisian endemic pemphigus foliaceus.

Pemphigus foliaceus (PF) is a bullous autoimmune skin disease diagnosed through sera and skin analyses. PF severity is associated with maintained anti-Dsg1 sera levels and its prognosis is unpredictable. MicroRNA (miRNA), dynamic regulators of immune function, have been identified as potential biomarkers for some autoimmune diseases. This study aimed to assess the miRNA expression of miR-17-5p, miR-21-5p, miR-146a-5p, miR-155-5p and miR-338-3p using quantitative real-time PCR in peripheral blood mononuclear cells (PBMC) and lesional skin samples from untreated and treated PF patients (both remittent and chronic) over 3 months. Overall, miRNA expression was significantly higher in PBMC than in biopsy samples. Blood miR-21 expression was increased in untreated patients compared to controls and had a diagnostic value with an AUC of 0.78. After 6 weeks, it decreased significantly, similar to anti-Dsg1 antibodies and the PDAI score. In addition, a positive correlation was observed between cutaneous miR-21 expression and the disease activity score. Conversely, cutaneous expressions of miR-17, miR-146a and miR-155 were significantly higher in treated chronic patients compared to remittent ones. The cutaneous level of miR-155 positively correlated with pemphigus activity, making it a potential predictive marker for patients' clinical stratification with an AUC of 0.86.These findings suggest that blood miR-21 and cutaneous miR-155 can be used as supplemental markers for PF diagnosis and activity, respectively in addition to classical parameters.

Biclonal Gammopathies in South Tunisia: Clinical and Biological Characteristics.

OBJECTIVE: Biclonal gammopathies (BGs) are rare situations characterized by the production of 2 monoclonal proteins. There are no available data on BGs in North Africa. We aimed to estimate the prevalence of BGs in our population and describe their clinical and laboratory features. METHODS: We conducted a 31-year retrospective study including patients with persistent double monoclonal bands based on the results of immunofixation/immunoelectrophoresis. RESULTS: A total of 35 patients with available clinical data (sex ratio, M/F = 1.53; mean age, 70 ±â€…10.87 years [range, 45-90 years]) were included. The main associated conditions were multiple myeloma (MM) (40%), BG of undetermined significance (BGUS) (34%), and lymphoproliferative diseases (23%). Only one-third of the patients had 2 monoclonal spikes on serum protein electrophoresis. The most common paraprotein combinations were immunoglobulin (Ig)G-IgG (25%) and IgG-IgA (23%) with different light chains in one-half of the cases. The mean follow-up was 25.6 months (median, 12 months). No BGUS evolved into a malignant disease. CONCLUSION: BGs are rare in clinical laboratory routine but must be accurately identified by the pathologist. Our cohort is characterized by a high prevalence of BGUS compared with MM.

Anti-mitochondrial antibodies detection assays for diagnosis of primary biliary cholangitis in southern Tunisia population / Techniques de détection des anticorps anti-mitochondries pour le diagnostic de cholangite biliaire primitive : étude dans une population sud-tunisienne

Anti-mitochondrial antibodies (AMA) represent serological markers of primary biliary cholangitis (PBC). Investigation of these autoantibodies can be performed by indirect immunofluorescence (IIF) on tissue sections or immunodot using M2 and M2-3E antigens. We aimed to evaluate the concordance of these immunological tests and their performance in PBC diagnosis. We reviewed sera which were tested for autoimmune liver disease anti-bodies by IIF (EUROIMMUN®) and immunodot (EUROIMMUN®). Results of IIF (AMA) and immunodot (anti-M2 and anti-M2-3E) were analyzed. A focus was given on positive results for AMA and/or anti-M2 and/or anti-M2-3E. According to available clinical data, patients were divided into two groups "PBC" and "Non PBC". Three-hundred-nineteen sera were tested by both techniques. Results of AMA, anti-M2 and anti-M2-3E were concordant in 296 cases (92.8%). Indeed, the three biomarkers were negative in 237 cases (74.3%) and positive in 59 cases (18.5%). Eighty-two sera were tested positive for AMA and/or anti-M2 and/or anti-M2-3E. Clinical data were available for 30 patients. In "PBC" group (n = 15), AMA, anti-M2 and anti-M2-3E antibodies were positive in 14/15 cases. PBC diagnosis was made in 12/15 patients without requiring liver biopsy. In "non PBC" group (n = 15), AMA, anti-M2 and antiM2-3E antibodies were positive in 9/15 cases. However, PBC diagnosis was not reached in the absence of other diagnostic criteria. IIF represents a first-line technique for AMA detection while immunodot is useful to confirm antigenic specificity in IIF-AMA positive cases. Anti-M2 and/or anti-M2-3E can be detected in some IIF-AMA negative cases. Interpretation of these tests'results relays mainly on clinical context.

Les anticorps anti-mitochondries (AAM) peuvent être recherchés par immunofluorescence indirecte (IFI) ou immunodot en utilisant les Ag M2 et M2-3E. Afin d'évaluer la concordance de ces tests et leur intérêt dans le diagnostic de cholangite biliaire primitive (CBP), nous avons comparé les résultats de recherche des AAM (IFI), anti-M2 et anti-M2-3E (immunodot) de 319 sérums. Selon les données cliniques disponibles, les patients avec au moins un marqueur positif ont été classés en deux groupes « CBP ¼ et « non CBP ¼. Les résultats des trois marqueurs étaient concordants dans 296 cas (92,8 %). Au moins un marqueur était positif dans 82 cas. Dans le groupe « CBP ¼ (n = 15), les trois marqueurs étaient positifs dans 14 cas. Dans 12 cas, le diagnostic était retenu sans recours à la biopsie hépatique. Dans le groupe « non CBP ¼ (n = 15), les trois marqueurs étaient positifs dans neuf cas, mais les autres critères de CBP n'étaient pas remplis. L'IFI demeure la technique de première intention pour la recherche des AAM ; l'immunodot permet de confirmer la spécificité antigénique. L'interprétation, notamment des cas discordants, repose surtout sur le contexte clinique.

Annexin A1 and its receptor gene polymorphisms in systemic lupus erythematosus in the Tunisian population.

BACKGROUND: An association between ANXA1, FPR1 and FPR2 gene polymorphisms and the patho-physiology of many human diseases was suggested by numerous studies. OBJECTIVE: Our study aimed to evaluate association between common polymorphisms in the 9q21.13 and 19q13.41 and susceptibility to systemic lupus erythematosus (SLE) in the Tunisian population. MATERIALS: We performed a case-control study on 107 Tunisian SLE patients and 122 healthy controls to explore 9 polymorphisms of the three studied genes: rs2811226 and rs3739959 (ANXA1), rs5030880, rs1042229, rs1461765570, rs17849971, rs867228 (FPR1), rs17694990 and rs11666254 (FPR2). RESULTS: Four polymorphisms were found to be linked with SLE susceptibility: rs3739959-ANXA1 > G and GG (p = 0.021, OR = 1.73 and p = 0.014, OR = 2.06 respectively), rs867228-FPR1 > TT (p = 0.014, OR = 4.59), rs11666254-FPR2 > GG (p = 0.019, OR = 8.34) and rs17694990-FPR2 > T (p = 0.05, OR = 1.506). In homogenous groups of SLE patients depending on clinical manifestations and serological results, previous associations were confirmed with a panoply of manifestations of lupus including lupus nephritis, malar rash, mouth ulceration and hypocomplementia. CONCLUSION: Our study showed an association between ANXA1 > rs3739959, FPR1 > rs867228, FPR2 > rs11666254, FPR2 > rs17694990 and SLE susceptibility. Our results also showed a strong association between the two ANXA1 studied SNPs and LN which allowed us to suggest these two SNPs as biomarkers of LN development in SLE. Further research is needed to understand by which mechanism the gene variants affect susceptibility to SLE. Key Points • Lupus erythematosus is an autoimmune disease in which a panoply of factors are implicated • Annexin A1 interaction with its receptors are suggested as a target in therapy of a panoply of human disease in particular cancers • The present results highlighted the implication of Annexin A1 and its receptors gene polymorphisms in the physiopathology of lupus, in particular in the involvement of renal and cutaneous lesions.

Clinical and serological profile of systemic sclerosis in Tunisia: A retrospective observational study.

OBJECTIVE: The Prevalence of systemic sclerosis (SSc) and different clinical subsets varies across the world. Few data have been published on SSc patients in North Africa. Our objective was to describe a SSc cohort in south of Tunisia and to compare clinical findings, disease subsets and antibodies with other international SSc populations. METHODS: In this retrospective observational study, Folders of patients with SSc seen in the internal medicine section of the Hedi Chaker Hospital between 2000 and 2013 were retrospectively analyzed. The diagnosis of SSc was retained according to ACR/EULAR 2013 criteria. Patients were classified into diffuse cutaneous SSc and limited cutaneous SSc subsets. Comparison with other cohorts was made based on published information. RESULTS: A higher female: male ratio (8:1) and a higher diffuse subset prevalence (82%) was found in this Tunisian cohort comparing with others. We also found a lower prevalence of calcinosis and anticentromere antibodies. Within each subset, diffuse cutaneous and limited cutaneous scleroderma clinical findings were similar with other systemic sclerosis populations except for a very low prevalence in renal crisis and pulmonary hypertension. Our results indicate overlap syndrome defined as scleroderma associated with others connective tissue disorder's is a relatively common condition. CONCLUSION: With slight variations, perhaps due to genetic, environmental or referral factors, SSc in this cohort appears to be similar to that described in other part of the world.

Genetic association and phenotypic correlation of TLR4 but not NOD2 variants with Tunisian inflammatory bowel disease.

OBJECTIVE: The common association between NOD2/CARD15 and TLR4 gene variants with inflammatory bowel disease (IBD) has not been replicated in all studies. We studied the polymorphism of these two genes in Tunisian patients with IBD. METHODS: Polymorphisms of NOD2 (R702W, G908R and L1007fs) and TLR4 (Asp299Gly and Thr399Ile) genes were analyzed in 106 patients with IBD (68 with ulcerative colitis [UC], 38 with Crohn's disease [CD]) and 160 healthy controls using polymerase chain reaction-restriction fragment length polymorphism. Genotypes and phenotypes were correlated. RESULTS: The mutated allele of TLR4-Thr399Ile was strongly associated with IBD (9.4% in IBD, 7.4% in UC and 13.2% in CD vs 2.5% in controls; P = 0.0004, 0.014 and 0.00006, respectively). Heterozygous genotypes were significantly more frequent in patients with IBD (17.0%), UC (14.7%) and CD (21.1%) than in controls (5.0%) (P = 0.0012, 0.012 and 0.001, respectively). Interestingly, the wild genotype was found to be protective (odds ratio 0.24). The mutated allele of TLR4-Asp299Gly was more frequent in controls (6.8%) than in patients with IBD (2.9%). A phenotypic correlation of Asp299Gly-AG genotype with arthritis in UC patients was detected (P = 0.003). Regarding the NOD2 gene, the common variations studied were not polymorphic and there was no genetic association with IBD. CONCLUSION: The TLR4-Thr399Ile variant was strongly associated with susceptibility to IBD, whereas TLR4-Asp299Gly seems to play a role in the clinical expression of UC. The rarity and non-association of NOD2 mutations with IBD may reveal a genetic characteristic of the population in our region.