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

EXTRACTION AND CHARACTERISATION OF BRUCELLA ABORTUS STRAIN RB51 ROUGH LIPOPOLYSACCHARIDE

Brucellosis is an important zoonotic disease with considerable impacts on human and animal health. Brucella abortus strain RB51 vaccine is used for prevention of bovine brucellosis in Iran. Due to strain roughness, available serological tests cannot detect vaccinated animals. Detection of serological responses to the vaccine is important to monitor accurate vaccination implementation. Rough lipopolysaccharide (RLPS) of RB51 strain was extracted and characterised to develop serological tests for diagnosis of vaccinated animals. RLPS was extracted using phenol-chloroform-petroleum ether and evaluated by limulus amebocyte lysate (LAL) assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and agar gel immunodiffusion (AGID). According to our results, the extracted RLPS caused positive reaction in LAL assay. In SDS-PAGE, a band with a molecular weight around 14 kDa was identified after specific staining using silver nitrate. Double AGID of the RLPS with a hyperimmune serum resulted in a precipitation line formation. Our study showed that the method can be successfully used to extract RLPS from Brucella abortus strain RB51 as confirmed by LAL assay, PAGE and AGID. Key words: brucellosis, RB51 vaccine, rough lipopolysaccharid

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

Disrtibution of blaTEM, blaSHV and blaCTX-M genes among ESBL-producing P. aeruginosa isolated from Qazvin and Tehran hospitals, Iran

Background. Pseudomonas aeruginosa is as an important opportunistic human pathogen, which  is associated with several clinical infections that are usually difficult to treat because of resistance to multiple antimicrobials. The production of extended-spectrum Ã-lactamases (ESBLs) is an important mechanism of Ã-lactam resistance. The aims of this study were to determine the prevalence of ESBLs, antimicroboial susceptibility, and to detect the blaTEM, blaSHV, and blaCTX-M genes.Methods. In this study, during March 2013  to December 2014, 266 P. aeruginosa isolates were collected from patients admitted to educational hospitals of Qazvin and Tehran, Iran. All isolates were initially screened for ESBL production by disk diffusion method and were further confirmed using a combined disk method. Antimicrobial susceptibility of ESBL-producing isolates was determined by standard disk diffusion method. PCR and sequencing techniques were employed for detection of blaTEM, blaSHV, and blaCTX-M genes.  Results.  In total, 262 (98.5%) P. aeruginosa isolates were non-susceptible to the used extended spectrum cephalosporins, among those 75(28.6%)  isolates were ESBL producers. Fifty nine (78.7%) of ESBL-producing isolates showed multidrug resistance pattern. Of 75 ESBL-positive isolates, the blaTEM-1 (26.7%) was the most common gene, followed by blaCTX-M-15 (17.3%), blaSHV-1 (6.7%), and blaSHV-12 (4%) either alone or in combination.Conclusions. The results of this study showed the notable prevalence of ESBLs among the clinical isolates of P. aeruginosa in Iran, indicating the urgency for implementation of appropriate follow-up measures for infection control and proper administration of antimicrobial agents in our medical settings