Prevalence of Clarithromycin-Resistant Helicobacter pylori in Patients With Chronic Tonsillitis by Allele-Specific Scorpion Real-Time Polymerase Chain Reaction Assay

Objectives/Hypothesis: To investigate the allelic prevalence of resistance to clarithromycin in the DNA of clinical isolates of Helicobacter pylori obtained from biopsy specimens of patients with chronic tonsillitis by Scorpion real-time polymerase chain reaction (PCR). Study Design: Pathologic specimens of patients with chronic tonsillitis were used for rapid urease test, and blocks of paraffin-embedded tonsillar tissue were used for McMullen staining, rapid urease test, and Scorpion real-time PCR test. Methods: A total of 103 biopsy samples were obtained from patients with chronic tonsillitis and examined for the presence of clarithromycin resistant H. pylori. Modified McMullen staining and rapid urease test were done on the all the samples. The DNA of specimens was extracted from the pathology blocks, and Scorpion real-time PCR was performed on a final volume of 25 lL. Results: Of 103 biopsy specimens, 22 samples were identified as infected by H. pylori, of which none were sensitive to clarithromycin. One had the A2143G genotype, and four had the A2142G genotype. Two had a mixed sensitive and the A2143G genotype, and five had a mixed sensitive and A2142G genotype. One strain had a mixed genotype of sensitive, A2143G, and A2142G. Conclusions: The reported rate of resistance to clarithromycin is of great variation among H. pylori strains isolated from specimens in different countries. Our study showed that the most prevalent genotypes in our H. pylori-positive specimens was A2142G followed by A2143G, which is different from reported results of allele-specific genotyping of H. pylori strains isolated from gastric biopsy and may be a result of cross-resistance to erythromycin and other macrolides. Key Words: Scorpion real-time polymerase chain reaction, chronic tonsillitis, clarithromycin

Identification of Legionella Pneumophila in Intubated Patients With TaqMan Real Time PCR

BACKGROUND: Legionellaceae contains Legionella genus with over 52 species and 64 serogroups. It is one of the most important causes of respiratory disease in human. More than 30% of hospital-acquired pneumonia is caused by Legionella. Ventilator-associated pneumonia (VAP) is an infection acquired in hospital wards, particularly in intensive care unit (ICU). This disease approximately affects 9% to 20% of intubated patients. Mortality in these patients varies between 8% and 76%. Legionella is one of the important factors for infection in intubated patients. OBJECTIVES: The present study was aimed to investigate the use of molecular methods in diagnosis of infection caused by Legionella pneumophila. MATERIALS AND METHODS: In this study, 109 samples of lung secretions collected from intubated patients admitted to ICU wards of four university hospitals in a three-month period were examined. Cultivation and Real time Polymerase Chain Reaction (PCR) methods were used to assess L. pneumophila colonization in these samples. RESULTS: Eleven samples had positive results using real time PCR analysis of 16s rRNA gene fragments specific for L. pneumophila, but according to culture method on specific buffered charcoal-yeast extract medium (BCYE), no positive cases were detected. Of the total positive cases, six were males, one female and four infants. The seven adults aged 40-65 years. CONCLUSIONS: Using molecular methods in diagnosis of infection caused by L. pneumophila has a great value because of its high specificity and rapid diagnosis potency

The Effects of Kainic Acid-Induced Seizure on Gene Expression of Brain Neurotransmitter Receptors in Mice Using RT2 PCR Array

Introduction: Kainic acid (KA) induces neuropathological changes in specific regions of the mouse hippocampus comparable to changes seen in patients with chronic temporal lobe epilepsy (TLE). According to different studies, the expression of a number of genes are altered in the adult rat hippocampus after status epilepticus (SE) induced by KA. This study aimed to quantitatively evaluate changes in the gene expression of brain neurotransmitter receptors one week after administration of kainic acid in the mouse hippocampus. Methods: We used 12 BALB/c mice in this study and randomly divided them into 2 groups. To both groups, saline (IP) was administered for 7 days, and on the last day, KA (10 mg/kg, IP) was injected 30 minutes after administration of saline. Subsequently, behavioural changes were observed in mice. Then, in one group (1 day group), 2 hours and in another group (7 days group), 7 days after KA administration, the hippocampus tissue of mice was removed and used for gene expression analyses. Total brain RNA was isolated and reversely transcribed. We performed qPCR using RT2 Profiler TMPCR Array Mouse Neurotransmitter Receptors and Regulators (QIAGEN) containing primers for 84 genes. In this regard, we selected 50 related genes for KA model. Results: Our results showed significant changes in the gene expression of GABAA subunits receptors, including α1-α3, α5, α6, β2, β3, γ1, ρ, and rho1-2 on day 7 compared with the day 1. Conclusion: Expression of both inhibitory and excitatory receptors changed after one week. Further studies are needed to find more molecular changes in the gene expression of brain neurotransmitter receptors and regulators over longer periods of time in KA models using RT2 PCR array

Capsid Modified Bluetongue Virus 16 (BTV16) as a Virulytic Oncotherapy Agent

Objective: Using potential viruses to destroy cancer cells has a long history, but recent advances in molecular biology raised hopes for successful use of these viruses again. Methods: Octreotate sequence was inserted into the neutralization region (R1& R2) in vp2 protein of capsid segment in 10 segmented genome of BTV in 304 - 368 position. T7 BTV RNA transcripts were extracted. Cancerous cultured cells were transfected with wild and modified BTV to recover BTV with cDNA-derived genome segments. Results: The results of all the performed experiments revealed that treatment of AGS cell lines with VP2 modified BTV16, which targeted cell surface of cancerous cells, significantly increased apoptosis in cancer infected cells. Conclusions: Modified VP2 BTV16 may be used as a potential virulytic oncotherapy agent in AGS cells. Keywords: Bluetongue Virus, Oncotherapy, Virulytic, AG

Mutation in alkylhydroperoxidase D gene dramatically decreases persistence of Mycobacterium bovis bacillus calmette-guerin in infected macrophage

Background and Objectives: Mycobacterium tuberculosis is the leading cause of death from a single bacterial species in the world and is subjected to a highly oxidative environment in its host macrophage and consequently has evolved protective mechanisms against reactive oxygen and nitrogen intermediates. Alkyl hydroperoxidase D (AhpD) is a molecule from these mycobacterial defense systems that has a dual function. It not only works with Alkyl hydroperoxidase C (AhpC) in mycobacterial defense system against oxidative stress but also has a role in oxidation/reduction of succinyltransferase B (SucB), dihydrolipoamide dehydrogenase (LPD) and AhpC. The present study was undertaken to find out the effects of inactivation of ahpD gene in the intra-macrophage persistence of resulted BCG mutant. Materials and Methods: We did allelic exchange mutagenesis in Mycobacterium bovis BCG and evaluate the effects of this mutagenesis in intracellular persistence of wild type BCG strains and ahpD mutant ones by comparing colony forming units (CFU) in infected macrophage. Results: Our findings showed that after producing allelic exchange mutagenesis in ahpD gene of M.bovis BCG a sever decrease in the CFU′s of ahpD mutant BCG strains has been observed and intracellular persistence of ahpD mutant BCG strains decreased significantly. Conclusion: Mutagenesis in ahpD gene will cause significant decrease in intracellular survival of ahpD mutant strains than wild type M.bovis BCG strains and could leads to an inefficiency in pyruvate dehydrogenase pathway and could also impair impairs mycobacterial defense system against oxidative and nitrosative stress