Morphological and Bactericidal Effects of Different Antibiotics on Helicobacter pylori

Background: Helicobacter pylori (H. pylori) is a spiral Gram negative bacteria that can transform to the coccoid form in adverse conditions. Objectives: The aim of this study was to determine the in vitro morphological and bactericidal effects of metronidazole, amoxicillin and clarithromycin on H. pylori. Materials and Methods: The standard strain 26695 of H. pylori was cultured on Brucella agar (BA) and the minimum inhibitory concentrations (MICs) of three antibiotics were determined by E-test method. The bacteria were exposed to antibiotics at 1/2 MIC, MIC and 2X MIC concentrations in Brucella broth (BB). Induced coccoid forms were confirmed by Gram staining and light microscopy. The viability of cells as well as the susceptibility of viable coccoids to antibiotics were examined using the flow cytometry method. Results: All of the three antibiotics at sub-MIC induced coccoid forms. The highest rates of coccoids (> 90%) were induced at 0.008 μg/ mL concentration (1/2 MIC) of amoxicillin, 72 hours postexposure. Metronidazole and clarithromycin with 1/2 MIC (0.5 and 0.125 µg/mL respectively) induced lower rates of coccoid forms (60% and 40% respectively). Potent bactericidal effects on coccoids were observed with Metronidazole at 2X MIC and clarithromycin at MIC (0.25 µg/mL) (80 - 90%). Amoxicillin with MIC and 2X MIC had no bactericidal effect on coccoid forms. Conclusions: Despite the good in vitro bactericidal effect of amoxicillin on spiral forms of H. pylori, this antibiotic has little effect on induced coccoids that may develop after the inappropriate in vivo antibacterial treatment. Hence, for successful therapy, it is essential not only to eradicate the spiral forms, but to eliminate the viable coccoids

Immunophenotypic subtyping of leukemic cells from Iranian patients with acute lymphoblastic leukaemia: Association to disease outcome

Background: Immunophenotypic characterization of the leukemic cells has been widely used as a tool for diagnosis, classification, stratification and prognosis of leukaemia. Objective: To investigate the immunophenotypic subtype profiles of Iranian patients with acute lymphoblastic leukemia (ALL) and its association to disease outcome. Methods: In this study, a total of 60 Iranian patients with ALL were immunophenotyped by flow cytometry using a panel of monoclonal antibodies specific for CD2, CD3, CD5, CD10, CD13, CD14, CD19, CD20, CD33, CD34, CD45, HLA-DR and TdT molecules. Results: The samples were initially categorized into T-ALL (n=9), B-ALL (n=50) and mixed lineage (n=1) based on the expression patterns of CD3 and CD19 molecules. B-ALL patients could further be classified into four subtypes, including Pro-B (n=7, 11.7), Pre-B I (n=28, 46.7), Pre-B II (n=13, 21.7) and immature/mature B cells (n=2, 3.3) on the basis of expression of CD10, CD19, CD20, HLA-DR and TdT. Clinical manifestations and laboratory findings of the patients did not reveal association with immunophenotypic subtypes of ALL, with the exception of mediastinal mass and WBC count at the time of diagnosis which were found to be significantly higher in patients with T-ALL compared with BALL (p=0.001 and 0.014), respectively. Conclusion: Our results indicate that overall the immunophenotypic profile of Iranian ALL patients is similar to previous reports and it might be used for monitoring of minimal residual disease and prognosis

Detection of anti-dsDNA by IgG ELISA test using two different sources of antigens: calf thymus versus E.coli

"nBackground: Anti-dsDNA antibodies frequently found in the sera Systemic Lupus Erythematosus patients, particularly in active disease stage. Nowadays exploit different eukaryotic and prokaryotic dsDNA as antigen source and different reagents as binder. The aim of this study to compared two dsDNA different sources and tow different kinds of reagents for binder in ELISA test. "nMethods: In this study bacterial genomic DNA from E.coli (ATCC 25922) and genomic DNA from calf thymus extracted with high purity and were used as antigens for IgG anti-dsDNA detection by ELISA. To coat dsDNA in microtiter wells, tow different kinds of reagents including methylated -BSA and poly-l-lysine (for pre-coating) are used. Sera from systemic lupus erythematosus patients and from normal blood donors are used to assess sensitivity and specificity of our ELISA test in compared with IF test and commercial kits. "nResults: Our results displayed pre-coating of microtiter plates with methylated -BSA reduce nonspecific binding reaction and the relative sensitivity and specificity of ELISA increased when calf thymus DNA is employed as antigenic source in compared with IF test and commercial kits 80%, 88% and 100%, 98% respectively, but when E.coli DNA is used 73%, 69% and 85%, 79%, respectively. "nConclusion: The genomic DNA from calf thymus is a potentially useful source of antigen for detection of anti-dsDNA by ELISA. Also the use of methylatted- BSA could have an effective role in reducing of nonspecific binding reactions

HLA-DRB, DQA and DQB allele frequencies in Iranian patients with chronic hepatitis B by PCR-SSP

Background: The outcome of acute hepatitis B infection may be influenced by host genetic factors like human leukocyte antigen (HLA). To investigate the association between the HLA-DRB, DQA1 and DQB1 alleles and chronic hepatitis B infection, 50 patients with chronic hepatitis B (based on 6 months positive of HBsAg and HBc antibody and HBeAg and antibody by serological test), were selected from Turkman population in north east of Iran .Allele frequency in patients were compared with a 65 aged and sex match control group from healthy blood donor of that ethnic population. Methods: HLA DRB, DQA1 and DQB1 alleles were determined using polymerase chain reaction based on sequence specific primer (PCR-SSP) method. Allele frequencies in patients and control subjects were compared by Epi-info statistical soft-wear. Results: There was a significant increase and positive association in HLA-DRB1*0301, DQA1*0501 and DQB1*0604 allele frequency in patients group while the frequency of HLA-DRB1*1301, 1501 and DQB1*0401 and DQA1*0401, 0102 were lower in patients than control group and shows negative association. Conclusion: In Iranian Torkman population, HLA DRB1*0301, DQA1*0501 and DQB1*0604 have an important role in susceptibility to chronic hepatitis B infection and HLA DRB1*1301, 1501, DQB1*0401 are associated with protection to chronic hepatitis B infection. Larger case control studies may be helpful to confirm our investigation

Assessment of Helicobacter pylori Viability by Flow Cytometry

Background: Flow cytometry is a rapid, sensitive, and reliable method for determination of bacterial viability. Here we assayed the capability of flow cytometry to detect Helicobacter pylori viable cells in both forms of spiral and coccoid. Methods: Viable bacteria stained with Rhodamin 123 and fluoresced with laser beam of 488nm. The rate of Rh123 absorption was determined in both forms of bacteria. Results: In positive control that consisted of live bacteria, the rate of rh123 absorption was at highest, but negative control that consisted of dead bacteria, the rate of Rh 123 absorption was at lowest absorption. This method showed that non-culturable coccoid forms of H. pylori, which could resist environmental stresses, were alive and might be responsible for bacterial transmission and failure in disease treatment. Conclusion: Due to simplicity, reliability, and sensitivity of flow cytometry, this method is preferred to other expensive and no reliable methods such as autoradiography, PCR and Electron microscopy used for assessment viability