Performance of existing diagnosis/classification criteria for Behcet's disease in Iranian patients: analysis of 5666 patients and 2406 controls

Aim: To analyse the performance of existing diagnosis/classification criteria for Behcet's disease (BD) in Iranian patients. There are 13 sets: Curth (1946), Hewitt (1969), Mason and Barnes (1969), Hewitt revised (1971), Japan (1972), Hubault and Hamza (1974), O'Duffy (1974), Cheng and Zhang (1980), Dilsen (1986), Japan revised (1988), International (1990), Iran (1993), Classification Tree (1993), and Dilsen revised criteria (2000). Methods: All patients from the Behcet's Disease Registry (5666) and control patients (2406) entered the study. Sensitivity, specificity and accuracy were calculated. Results: The most sensitive was Curth criteria with (99.7%), followed by Classification Tree (97.3%), Zhang (93.5%), Iran (91.4%), Japan revised (86.4%), Japan (85.3%), Dilsen (83.7%), Hubault and Hamza (81.6%), Dilsen revised (81.2%), International criteria (79.8%), Hewitt (73.8%), O'Duffy (70.7%), and Masson and Barnes (65.7%). The most specific was Masson and Barnes (99.6%), followed by the International criteria (98.3%), Dilsen revised (98.2%), O'Duffy (97.6%), Japan (97.1%), Japan revised (97%), Classification Tree (96.7%), Hewitt (95.8%), Iran (95.8%), Zhang (92.4%), Dilsen (91.4%), Hubault (90.8%), and Curth (78.6%). The most accurate criteria was Classification Tree (97.1%), followed by Curth (93.4%), Zhang (93.1%), Iran (92.7%), Japan revised (89.6%), Japan (88.8%), Dilsen revised (86.2%), Dilsen (86%), International criteria (85.3%), Hubault (84.3%), Hewitt (80.4%), O'Duffy (78.8%), and Mason and Barnes (75.8%). Discussion: Among the existing criteria, the best to classify Iranian patients is the Classification Tree. The second most accurate is Curth criteria. The difference is statistically significant. Further, Curth criteria is not optimized, having very high sensitivity and low specificity.     

The effects of ranitidine and cimetidine on the pharmacokinetics and pharmacodynamics of metoprolol

The impact of cimetidine, ranitidine and placebo on the pharmacokinetics of metoprolol, given either as a single dose (100 mg) or for 7 days (100 mg b.d.), has been evaluated in two separate studies. The doses used were 800 mg cimetidine daily and 300 mg ranitidine daily. The subjects were all young, healthy volunteers. In the single dose study, cimetidine produced a marked increase in the peak plasma concentration of metoprolol and in the area under the plasma concentration-time curve; ranitidine had less effect, though the area under the curve was significantly greater than placebo. In the chronic dosing study, the area under the curve for metoprolol was also significantly higher on cimetidine (1796 ng h/ml; P less than 0.001) whereas the area under the curve on ranitidine (1258 ng h/ml) was comparable to that on placebo (1183 ng h/ml). Despite these drug-induced changes in plasma metoprolol concentration, neither cimetidine nor ranitidine altered the change in exercise-induced heart rate during dosing with metoprolol.     

Conjugated Linoleic Acid Stimulates Apoptosis in RH and Tehran Strains of Toxoplasma gondii,in Vitro

Background:

The aim of this study was to evaluate the effects of conjugated linoleic acid (CLA) on apoptosis of tachyzoites of T. gondii, RH strain (type I) and the cyst-forming Tehran strain (type II) in vitro.

Methods:

Toxoplasma strains were injected into the peritoneal cavity of BALB/c mice. The Tehran strain forms cysts in the brain of mice. Bradyzoites within the cysts are reactivated to proliferative tachyzoites, by dexamethasone. Tachyzoites were aspirated from the peritoneum of infected mice, and the percentage of viable parasites was estimated with trypan blue staining. Tachyzoites were inoculated into HeLa cells cultivated in DMEM medium. Different concentrations of CLA were evaluated on T. gondii in HeLa cells by the tetrazolium (MTT) colorimetric assay. Differentiation between apoptosis and cell death was determined by flow cytometry using Annexin V and propidium iodide (PI) double staining. The statistical analysis performed by GraphPad Prism version 6.00.

Results:

CLA induces apoptosis in virulent (RH) and avirulent (Tehran) strains of T. gondii. The results of MTT indicated that CLA could decrease the proliferation of tachyzoites of both strains in HeLa cells.

Conclusion:

Conjugated linoleic acid has anti-toxoplasmacidal activity on tachyzoites of T. gondii. Therefore, we recommended further studies on this component in order to achieve a new drug against the parasite.     

Rituximab in intractable ocular lesions of Behcet’s disease; randomized single‐blind control study (pilot study)

Background: Ocular lesions, the main morbidity of Behcet’s disease (BD), are the most difficult to treat. The aim of this study was to evaluate the efficacy of rituximab. Methods: Inclusion criteria were retinal vasculitis and edema, resistant to cytotoxic drugs. Twenty patients were randomized to a rituximab group (RG) or cytotoxic combination therapy group (CCTG). Rituximab was given in two 1000‐mg courses (15‐day interval). Subjects received methotrexate (15 mg/weekly) with prednisolone (0.5 mg/kg per day). The CCTG received pulse cyclophosphamide (1000 mg/monthly), azathioprine (2–3 mg/kg per day) and prednisolone (0.5 mg/kg per day). The primary endpoint was the overall state of patients’ eyes and the Total Adjusted Disease Activity Index (TADAI). Secondary endpoints were: visual acuity (VA), posterior uveitis (PU), and retinal vasculitis (RV). The baseline data were compared at 6 months by paired sample t‐test and analysis of variance. Results: TADAI improved significantly in the RG (t = 3.340, P = 0.009), but not in the CCTG (t = 2.241, P = 0.052). For secondary endpoints (RG/CCTG), the mean VA improved in two patients versus three (2/3), remained unchanged in 1/1, and worsened in 7/6 patients. The mean PU improved significantly in the RG (t = 3.943, P = 0.001), not in the CCTG (t = 2.371, P = 0.028). RV improved, but not statistically (t = 2.027, P = 0.057 vs. t = 1.045, P = 0.31). Edema of retina, disc and macula improved significantly in both, but much better for the RG (t = 2.781, P = 0.012 vs. t = 2.707, P = 0.014). Conclusion: Rituximab was efficient in severe ocular manifestations of BD, TADAI improved significantly after 6 months with rituximab, but not with CCT.     

Validation of the International Criteria for Behçet’s disease (ICBD) in Iran

Aim: To evaluate the performance of the new International Criteria for Behçet’s Disease (ICBD) in Iran. The ICBD was created in 2006 to replace the International Study Group (ISG) criteria (created in 1990). In this study, 14 existing diagnosis/classification criteria (Curth, Hewitt, Mason and Barnes, Japan original, Hubault and Hamza, O’Duffy, Cheng and Zhang, Dilsen, Japan revised, International Study Group, Iran traditional, Iran Classification Tree, revised Dilsen, Korea), were evaluated and compared to ICBD by calculating their sensitivity, specificity and accuracy. Methods: All patients from the Behçet’s Disease registry (6128) and controls (3400), up to January 2008, entered the study. The diagnosis was clinical and not by any of the above‐mentioned criteria. Sensitivity, specificity, accuracy (percent agreement), and 95% confidence interval were calculated. The performance of ICBD and ISG were compared by the McNemar test. Results: The sensitivity was 98.2% for ICBD and 78.1% for ISG (P < 0.0001). The specificity was 95.6% for ICBD and 98.4% for ISG (P < 0.0001). The accuracy was 97.3% for ICBD and 85.5% for ISG (P < 0.0001). Discussion: In Iranian patients, ICBD has 20.1% higher sensitivity and 11.8% higher accuracy than ISG, while the specificity was only 2.8% lower. ICBD was better optimized than ISG (difference between sensitivity and specificity 2.6% vs 20.3%). Conclusion: ICBD has by far better performance than ISG in Iran. The difference is even more prominent in Iranian patients than in the cohort of the international patients upon whom the criteria were created.     

Triglyceride and high‐density lipoprotein levels as the markers of disease activity and their association with TNF‐α and TNF receptor system in systemic lupus erythematosus

Objective: There are some clues that measurement of triglyceride (TG) and high‐density lipoprotein (HDL) and their correlation with tumor necrosis factor alpha (TNF‐α), soluble TNF‐α receptor type 1 and type 2 (sTNFR1, sTNFR2) in serum could be valuable in the assessment of disease activity of systemic lupus erythematosus (SLE) patients. Methods: In this cross‐sectional study, fasting blood samples were obtained from 86 SLE patients referred to the Rheumatology Research Center of Tehran University in Shariati Hospital, Iran. Disease activity was determined by using the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). TG and HDL obtained after overnight fasting were analysed by routine chemistry. Levels of circulating TNF‐α, sTNFR1, and sTNFR2 were determined by enzyme‐linked immunosorbent assay. Results: Triglyceride levels were associated with SLEDAI (r = 0.59, P < 0.001), TNF‐α (r = 0.27, P < 0.01), and with sTNFR1 (r = 0.54, P < 0.001); on the contrary, HDL levels were negatively associated with SLEDAI (r = –0.29, P < 0.007), TNF‐α (r = –0.27, P < 0.01), and sTNFR1 (r = –0.35, P < 0.001). The correlation of TG and HDL with sTNFR2 were (r = 0.13, P > 0.23) and (r = –0.17, P > 0.1), respectively. In multiple logistic regression models, the levels of TNF‐α and HDL were omitted. Conclusion: Dyslipoproteinemia with high TG/low HDL levels correlates with disease activity in SLE, and enhanced activity in the TNF‐α/sTNFR system. With results of this study and the same studies, serum levels of TG, HDL, TNF‐α, sTNFR1, sTNFR2 are valuable markers for estimation of disease activity in SLE.     

Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran

Background

Parasitological methods for the diagnosis of Visceral leishmaniasis (VL) require invasive procedures, so serological and molecular approaches have been developed but are not generally applicable in the field. We evaluated a loop mediated isothermal amplification (LAMP) assay using blood from VL patients and compared it to nested PCR.

Methods

Forty-seven subjects with clinical features (fever, hepatosplenomegaly and anemia) were confirmed positive for VL by the direct agglutination test (DAT) at titers >3200. Forty DAT negative individuals from non-endemic areas with no clinical signs or symptoms of VL served as controls. A LAMP assay was performed using a set of six primers targeting Leishmania infantum kinetoplast DNA (kDNA) minicircle gene under isothermal (64 °C) conditions. For nested PCR we used primers targeting the kDNA minicircle gene.

Results

The LAMP assay provided a detection limit of 1 parasite in 1 ml of peripheral blood and detected L. infantum DNA in 44 of 47 DAT-confirmed VL cases, with diagnostic sensitivity of 93.6% (95% CI). No L. infantum DNA was amplified in controls, indicating a specificity of 100%. The nested PCR yielded sensitivity of 96% (95% CI) and a specificity of 100% (95% CI).

Conclusion

The LAMP assay gave results similar to those of nested PCR but in a shorter time. The LAMP method is simple; requires no sophisticated equipment; has a short reaction time; and results, indicated by turbidity of the reaction mixture, are observable with the naked eye.     

Pathergy test in Behcet's disease: change in incidence over the time

Introduction: Pathergy phenomenon (PP) is used as a criterion in many diagnostic criteria for Behcet's disease (BD): Curth, Japan, Hubault and Hamza, Cheng and Zhang, Dilsen, International Study Group, Iran criteria (traditional format and the Classification Tree), Korea criteria, and the new International Criteria. PP is rather specific to BD. It has been reported with high frequency from most countries along the Silk Road, but with much lower frequency from other countries (UK, US). The incidence of PP has been reported from some countries (Japan, Turkey) to decrease gradually. The aim of this study was to test the above hypothesis in a large cohort of patients in Iran. Materials: Patients (6057) were divided into several groups by two methods. Method 1: patients were divided into six groups according to the time of their first visit. Method 2: patients were divided into four groups according to the date of the beginning of their first manifestation. Results: In method 1, the percentage of PP was 71.8% from patients 1–1000. The percentage became 55.9% from patients 1001–2000, 57.6% from patients 2001–3000, 58.8% from patients 3001–4000, 45.3% from patients 4001–5000, and 33.9% from patients 5001–6000. In method 2, the percentage of PP was 61.5% for patients with their disease onset before 1977. The percentage was 59.9% for patients with their disease starting between 1978 and 1987. The percentage dropped to 52.1% for patients with disease onset between 1988 and 1997. The percentage finally dropped to 41% for patients having their disease onset between 1998 and 2007. Conclusion: The incidence of PP is decreasing gradually. It is now 34%, which is rather low for a criterion used in many diagnosis criteria. It is important to find a surrogate for it because with the decrease in the incidence of PP the sensitivity of the diagnosis criteria will drop significantly.     

Management of ocular manifestations of Behcet's disease: outcome with cytotoxic drugs

Background and aim: Cytotoxic drugs, combined with steroids, are the first line of treatment for serious ocular manifestations in Behcet's disease (BD) such as uveitis and retinal vasculitis. If used judiciously, they are effective and safe, even in the long‐term. In our unit, the following cytotoxic drugs have been used for this purpose – oral or pulse cyclophosphamide, weekly methotrexate, chlorambucil, cyclosporin, azathioprine, or a combination of these. In our experience, these cytotoxic drugs had a similar range of efficacy regarding improvement in visual acuity. Having obtained the same treatment response, we pooled all our data together to evaluate the overall outcome of the eye complications in our cohort of BD patients. Methods: The treatment protocol was the same regardless of which cytotoxic drug was used. Prednisolone was given to all patients at an initial dose of 0.5 mg per kilogram of body weight per day. The dose was later tapered as necessary. If a patient was resistant to a given cytotoxic drug, the drug was changed to another. In pooled data, the results before the first treatment were compared with those after the last treatment. Visual acuity (VA) was measured using a Snellen chart (on a scale of 10). The Disease Activity Index (DAI) for anterior uveitis (AU), posterior uveitis (PU), and retinal vasculitis (RV) was measured according to the criteria of Ben Ezra. The Total Inflammatory Activity Index (TIAI) and the Total Adjusted Disease Activity Index (TADAI) were also calculated. Statistical analysis was performed using a Student's t‐test for paired samples comparing the data obtained before and after treatment. Results: At the last evaluation of our database (March 2004) on patients who had an active posterior uveitis and/or retinal vasculitis, 1104 were treated with cytotoxic drugs, of whom 290 patients received more than one treatment protocol. The mean duration of eye lesions was 32.8 months (SD 37.6). The mean treatment time was 20.1 months (SD 20.3). Overall, the VA of 70% of patients improved or maintained after treatment. The mean VA improved from 3.8 to 4.7 (t = 10.099, P < 0.000001). The mean DAI of AU improved from 2.5 to 0.8 (t = 19.575, P < 0.000001). The mean DAI of posterior uveitis improved from 2.1 to 0.9 (t = 28.616, P < 0.000001). The mean DAI of retinal vasculitis improved from 2.5 to 1.5 (t = 12.070, P < 0.000001). The mean TIAI improved from 16.5 to 7.9 (t = 20.13, P < 0.000001). The mean TADAI improved from 37.1 to 25.6 (t = 20.24, P < 0.000001). Further analysis showed methotrexate at a dose of 15 mg/week, and azathioprine at 2 mg/kg/day were preferred for posterior uveitis, while low dose pulsed cyclophosphamide (0.5 g/m²/month) – azathioprine combination therapy for retinal vasculitis. Conclusion: Cytotoxic drugs are efficacious in the treatment of serious ocular manifestations of BD.