Typing methods used in the molecular epidemiology of microbial pathogens: a how-to guide.

Microbial typing is often employed to determine the source and routes of infections, confirm or rule out outbreaks, trace cross-transmission of healthcare-associated pathogens, recognize virulent strains and evaluate the effectiveness of control measures. Conventional microbial typing methods have occasionally been useful in describing the epidemiology of infectious diseases. However, these methods are generally considered too variable, labour intensive and time-consuming to be of practical value in epidemiological investigations. Moreover, these approaches have proved to be insufficiently discriminatory and poorly reproducible. DNA-based typing methods rely on the analysis of the genetic material of a microorganism. In recent years, several methods have been introduced and developed for investigation of the molecular epidemiology of microbial pathogens. Each of them has advantages and limitations that make them useful in some studies and restrictive in others. The choice of a molecular typing method therefore will depend on the skill level and resources of the laboratory and the aim and scale of the investigation. This study reviews the most popular DNA-based molecular typing methods used in the epidemiology of bacterial pathogens together with their advantages and limitations.

Novel cassette array in a class 1 integron in clinical isolates of Acinetobacter baumannii from central Iran.

Antibiotic resistance in Acinetobacter baumannii is a major problem in the hospital and outbreaks caused by this organism have been reported frequently. The present study aimed at determining the antibiotic susceptibility patterns, the prevalence of different classes of integrons and the characterization of integron class 1 gene cassettes in Iranian A. baumannii isolates. A total of 63 non-duplicate A. baumannii isolates were collected from clinical and environmental specimens in the Vali-Asr hospital in the central province of Iran (March to September, 2011). The antimicrobial susceptibility for 15 antibiotics which are used conventionally was determined by disk diffusion. The presence of different integron classes was investigated by PCR and the size of gene cassettes in class 1 integrons was then determined by PCR as well. Moreover, integron cassette arrays of isolates were delineated by RFLP and sequencing amplicons with different lengths. Of 63 isolates 62 (98.4%) carried a class 1 integron. The prevalence of IntI2 was 15.9% and the length of the amplicons ranged from 500 bp to 3 kb. Sequencing of integrons of class 1 revealed the presence of many resistance genes (aadA, aacA, aacC, dfrA, bla(GES) and bla(IMP)). We identified a completely new gene cassette which contained aacA7-qacF-aadA5-bla(IMP), this cassette has not been reported previously in A. baumannii.

New immunological investigations on Helicobacter pylori-induced gastric ulcer in patients.

Although Helicobacter pylori (Hp) plays an important role in the pathogenesis of chronic gastritis and gastric ulcer, little is known about the probable mechanisms of these types of gastrointestinal damage. To determine the precise mechanisms involved in ulcer formation, immune responses in patients with gastric ulcer (GUP) caused by Hp infection (Hp(+)) were compared with those of other gastritis patients (GP). The sensitivity and proliferation of peripheral blood mononuclear cells (PBMNCs) obtained from patients were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against exposure with complex Hp crude antigen (HPCA) and mitogen (phytohemagglutinin, PHA). Production of inflammatory cytokines, including interleukin (IL)-1ß and IL-8, in serum and supernatants of PBMNCs were then measured by ELISA. It was found that, after stimulation with PHA, both IL-8 and IL-1ß concentrations in sera and supernatants as well as proliferation and sensitivity were statistically greater in GUP Hp(+) than GP Hp(-) . Furthermore, HPCA inhibited the proliferation of PBMNCs dose-dependently; however, it stimulated IL-8 and IL-1ß production in supernatants of mononuclear cells. Therefore, the up-regulated concentrations of IL-8 and IL-1ß may have been caused by increase in the size of mononuclear cell subpopulations or in their cytokine secretory activity, indicating the greatest cell responsiveness in GUP Hp(+) patients. These results suggest that tissue damage and ulcers occur in patients who produce more IL-8 and IL-1ß than patients who do not develop ulcers; the former consequently have more activated immune cells at the site of infection. Therefore, both host responses and Hp virulence factors may be involved in the development of gastric ulcers.

Validation of an in-house made rapid urease test kit against the commercial CLO-test in detecting Helicobacter pylori infection in the patients with gastric disorders.

BACKGROUND: H. pylori is a urease positive organism, and this activity in a gastric biopsy could be considered as a proof of the presence of H. pylori. For the reasons of high price and difficult accessibility to the commercial CLO-test in Iran, we designed an affordable equivalent test with high specificity, accuracy and availability. METHODS: Biopsy samples from 80 symptomatic patients with gastrointestinal problems were included in this study. The results of our in-house made rapid urease kit were compared with the commercial CLO-test up to 3 hours and 24 hours after inoculation of the biopsy samples. Culture results and gram staining were proposed as gold standard. RESULTS: Helicobacter pylori was isolated from 36 patients (45.0%) after cultivation of biopsy samples. After 3 hours, 33 (91.6%) cases of positive samples for H. pylori, showed urease positive reaction using both, in-house made and CLO-test kits. However, 2 (5.5%) cases showed urease reaction at 24 hours using both the kits. The specificity of 100% was determined for both, in-house made and commercial CLO-test kits after 3 hours. The sensitivity for both the kits was estimated at 97.1% after 3hours. However, after 24 hours, sensitivity and specificity of 97.1% and 88.64% was estimated for the in-house and 97.2 % and 95.4% for the commercial CLO-test kits, respectively. CONCLUSION: Specificity and sensitivity of 100% and 97.1 % for up to 3 hours follow biopsy sampling, could be considered as an advantage for our in-house rapid urease kit. Moreover, the rapid urease agar media designed in our lab is cost-effective with adequate sensitivity and specificity levels for the detection of H. pylori, compared with the commercial CLO-test.

Serotypes, antibiotic resistance, and class 1 integrons in Salmonella isolates from pediatric cases of enteritis in Tehran, Iran.

The present study was conducted to investigate serotype distribution, antimicrobial resistance patterns, carriage of class 1 integron, and clonality of Salmonella strains isolated from patients aged 0-12 years in Tehran, Iran, during 2007-2008. A total of 139 Salmonella isolates were studied. Salmonella serotypes Enteritidis, Infantis, and Typhimurium included 84.9% of isolates, Enteritidis accounting for 41.7%. The most prevalent resistances were to doxycycline (64.7%), nalidixic acid (61.2%), tetracycline (51.8%), and streptomycin (42.8%). Fifty-three (38.1%) isolates contained class 1 integron. Eight different gene cassettes were identified, aadA1 being the most frequently encountered. Pulsed-field gel electrophoresis showed that integron-positive Salmonella strains belonging to serotypes Infantis, Enteritidis, and Typhimurium were attributed to two, three, and five different pulsotypes, respectively. The findings indicated that the distribution and drug resistance pattern of most prevalent Salmonella serotypes were broadly similar to that reported globally from human isolates. Presence of class 1 integrons was common among Salmonella serotypes in Tehran, Iran. Concurrent clonal expansion and horizontal transmission events seem to contribute to increase in drug resistance prevalence among Salmonella serotypes.

An improvement in isolation and preservation of clinical strains of Helicobacter pylori.

BACKGROUND AND AIM: Isolation of H. pylori from gastric mucosal biopsy specimens is a prerequisite for further studies addressing drug susceptibility testing, analysis and characterization of virulence factors, molecular epidemiology studying or other comparative studies. In this study, we used a modified enriched culture medium with short incubation time to improve the isolation rate of H. pylori from the clinical specimens. METHODS: Between October 2008 and October 2009, 266 dyspeptic patients attending the endoscopy ward of Motahhary Clinic of Shiraz University of Medical Sciences, were investigated. The biopsy samples were cultured on two selective media called M1, which we used in our previous studies, and a modified medium called M2. The cultures were kept in a microaerophilic atmosphere at 37 degrees C. The plates were inspected first on day 1, and then on daily basis for a total of 10 days. The isolates were confirmed as H. pylori by colony morphology and positive oxidase, catalase and rapid urease tests. We used the same media and culture conditions to subculture the isolates for several times. Specimens were considered to be H. pylori positive if either the culture or two of the three diagnostic methods yielded positive results. RESULTS: The isolation rate of H. pylori strains from the samples was significantly higher on M2 in comparison with M1 medium (p<0.05). The bacterial growth on M2 was observed after a significantly shorter time (p<0.05), i.e., after incubation for about 24 hrs. Following these procedures, the preservation time could be extended beyond 6 months without a significant loss of viability. CONCLUSION: The modified culture technique enabled a shorter incubation time and a higher isolation rate for H.pylori obtained from clinical samples.

Antibacterial susceptibility patterns and cross-resistance of methicillin resistant and sensitive staphyloccus aureus isolated from the hospitalized patients in shiraz, iran.

Nosocomial infections caused by methicillin-resistant staphylococci (MRSA) pose a serious problem in many countries. This study aimed to determine the antibacterial susceptibility patterns of methicillin sensitive and resistant Staphylococcus aureus isolates from the hospitalized patients. Totally 356 isolates of Staphylococcus aureus (S. aureus) including 200, 137 and 19 corresponding to MSSA, MRSA, and intermediate MRSA strains, respectively were isolated. Antibacterial susceptibility patterns of the isolates to 14 antibiotics were examined using Kirby-Bauer method. MICs of 15 antibiotics to 156 MRSA isolates were determined by E test method. Cross-resistances of MRSA isolates (137+19) to the other tested antibiotics were also determined. S.aureus with high frequencies were isolated from the blood, sputum and deep wound samples. All of 200 MSSA isolates were sensitive to oxacillin, vancomycin, tecoplanin, rifampin, linezolid, quinupristin/dalfopristin, mupirocin and fusidic acid. A gradient of reduced susceptibility of MSSA to cephalexin, co-trimoxazole, ciprofloxacin, clindamycin, tetracycline, erythromycin and gentamicin were evident. MRSA isolates were sensitive to vancomycin, tecoplanin, linezolid, quinupristin/dalfopristin, mupirocin and fusidic acid, while reduced susceptibility of them to rifampin, co-trimoxazole, clindamycin, cephalexin, tetracycline, ciprofloxacin, erythromycin and gentamicin were observed. MRSA isolates exhibited a high range of cross-resistance to the eight tested antibiotics. Overall, co-trimoxazole, ciprofloxacin, clindamycin, tetracycline, erythromycin and gentamicin showed low activity against MSSA and MRSA isolates which may indicate they are not suitable to be used in clinical practices. To preserve the effectiveness of antibiotics, rational prescription and concomitant application of preventive measures against the spread of MRSA are recommended.

Assay for integrons and pattern of antibiotic resistance in clinical Escherichia coli strains by PCR-RFLP in Southern Iran.

The purpose of this study was to determine the prevalence of multidrug-resistant Escherichia coli in clinical specimens. In addition, the existence of integrons in resistant isolates was assessed by amplification of intergase genes. Susceptibility of 200 isolates from five Shiraz hospitals and health centers to 13 antibiotics was determined by the Kirby-Bauer disk diffusion method. The majority of the bacteria were isolated from urine (70.5%) and stool (25.5%) specimens. Antibiotic resistance patterns were observed as follows: amoxicillin 63%, tetracycline 57.5%, co-trimoxazole 48%, cephalotin 40%, nalidixic acid 36%, ciprofloxacin 21%, nitrofurantoin 25%, norfloxacin 20.5%, gentamicin 18%, chloramphenicol 18%, ceftazidime 14%, amikacin 8.5% and imipenem 2%. Of 200 isolates tested, 165 (82.5%) were multidrug resistant. The frequency of multidrug resistance to more than 5 antibiotics was 24.2%. The existence of integrons was confirmed in 44.8% of isolates. Significant association between resistance to gentamicin, amikacin, cephalotin, nalidixic acid, ciprofloxacin, norfloxacin and co-trimoxazole with the existence of integrons was obtained by the PCR-RFLP method. These results showed that integrons may be partly responsible for multidrug resistance. Imipenem, amikacin and ceftazidime were the most effective antibiotics in vitro; however, the clinical efficacy of these antibiotics remains to be assessed.

Is Escherichia coli O157:H7 a common pathogen in children with bloody diarrhea in Shiraz, Iran?

Escherichia coli (E. coli) O157:H7 is a common cause of bloody diarrhea in developed countries. The aim of this study was to determine whether E. coli O157:H7 is a possible pathogen of bloody diarrhea in southern Iran. Out of 719 children with diarrhea, 243 (34%) patients with positive occult blood took part in our study. The polyclonal antibody test and polymerase chain reaction (PCR) were used to identify E. coli O157:H7. Stool cultures showed enteropathogens in 107 patients (44%). Shigella (34.3%) was followed by E. coli (8.6%), campylobacter (2%) and salmonella (0.4%). None of the E. coli species was of O157:H7 serotype. Antibiotic sensitivity of shigella species was 100% to ceftriaxone, ciprofloxacin and ceftazidime, 94% to nalidixic acid and 13% to co-trimoxazole. The results of the study showed that E. coli O157:H7 is not a cause of bloody diarrhea in our area.

Low distribution of integrons among multidrug resistant E. coli strains isolated from children with community-acquired urinary tract infections in Shiraz, Iran.

Although integrons by themselves are not mobile, due to their presence in plasmids and transposons, they can be transferred horizontally. For these reasons integrons are a major mechanism for the spread and maintenance of multidrug resistance (MDR). This study describes the distribution of integron gene cassette classes in a collection of uropathogenic Escherichia coli (UPEC) isolated from children with community acquired urinary tract infection in Jahrom, Iran. E. coli strains isolated from urine samples were tested for susceptibility to 14 different antibiotics using the disk diffusion method and for integron classes by RFLP-PCR. Totally 96 strains of E. coli were isolated from urine samples. High prevalence of resistance to ampicillin (80.2%), co-trimoxazole ((76%) and tetracycline (70.8%) was seen among the UPEC isolates. All isolates were 100% sensitive to imipenem. Sixteen strains (16.6%) had the evidence ofintegron sequences with the prevalence of 6.25% (n = 6) and 10.41% (n = 10) for intI1 and intI2, respectively. No intI3 was detected in the isolates. The presence of integrons was significantly associated with resistance to certain antibiotics including gentamicin and ampicillin. Considering the MDR patterns and the low prevalence ofintegrons among the E. coli strains under the study, we suggest that the antibiotic resistance cassettes in these strains presumably are mostly carried on the other transposable elements rather than integrons.

Restriction fragment length polymorphism of virulence genes cagA, vacA and ureAB of Helicobacter pylori strains isolated from Iranian patients with gastric ulcer and nonulcer disease.

OBJECTIVE: To investigate the distribution of different genotypes of major virulence factors cagA, vacA and ureAB among Helicobacter pylori (H. pylori) strains isolated from patients with ulcerative and nonulcerative diseases. METHODS: This study was performed in Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran, during November 2004 to October 2005. Sixty-five H. pylori strains, 30 from patients with gastric ulcer (ulcerative disease) and 35 from patients with gastritis (nonulcerative disease) were analyzed by polymerase chain reaction (PCR) to investigate the presence of cagA, vacA and ureAB genes. The amplified fragments were then digested with the restriction enzymes HaeIII (for ureAB) HinfI (for cagA) and HphI (for vacA). RESULTS: We found a significantly higher prevalence of vacA-positive strains in ulcerative disease (UD) than that in nonulcerative disease (NUD) patients (p<0.05). Restriction fragment length polymorphism (RFLP) analysis revealed 2 different patterns for cagA gene. The prevalence of pattern beta with 3 bands was significantly higher in both groups of patients. HaeIII digestion resulted in a strictly homogeneous pattern for 83.33% of the vacA+ strains isolated from the patients with UD. This pattern was significantly associated with UD status (p<0.05). The ureAB polymorphism analysis revealed 10 distinguishable DNA banding patterns among them the pattern named ureAB 5a was the most prevalent (47.61%) in all isolates. No association between a specific DNA pattern and clinical disease was observed for cagA and ureAB (p>0.05). CONCLUSION: It seems that in our patients, the presence of cagA gene may not necessarily be a risk factor for ulcer disease, while a homologous genotype of vacA appears to be associated with an increase risk of UD development. Lastly, despite the existence of a high degree of genomic variability within ureAB, conserved DNA banding profiles are distributed in our areas.

Comparison of arbitrarily primed-polymerase chain reaction and plasmid profiles typing of Pseudomonas aeruginosa strains from burn patients and hospital environment.

OBJECTIVE: To identify the strengths and weakness of arbitrary primed-polymerase chain reaction (AP-PCR) and plasmid profiles for typing of Pseudomonas aeruginosa (P. aeruginosa) and tracking of source of infections. METHODS: Seventy-four strains of P. aeruginosa were isolated from burn patients and hospital environment between January to April 2003 in Ghotbadden Burn Hospital, Shiraz, Iran. The strains were classified by photo Capt Mw program, similarity and clustering of strains were assessed using NTSYS-PC version 2.02K software. RESULTS: Based on 50% and 64.7% and 67.5% similarity on the plotted dendrogram, 38 plasmid profiles were classified into: 2, 3 and 5 clusters, respectively. Photo Capt Mw program categorized AP-PCR products to 47 different types of 6 to 12 bands between 0.376 to 3.7 kb. Based on dendrogram pattern 3 levels (62 %, 81% and 84.6%) of similarity were selected. Using these criteria 2, 5 and 11 clusters were obtained, respectively. CONCLUSION: As compared with plasmid profiles, AP-PCR analysis protocol is rapid, reproducible and differentiated the isolates with higher discrimination power. These results suggest that during admission of patients in burn center a limited number of common strains cross-contaminate burn victims. However, transmissions of infection from hospital environment to patients also occur in the minority of the victims. To control cross-contamination of the patient wounds with antibiotics resistant isolates, strong disinfection of patients' bathroom after scrubbing of each patient wounds is mandatory.

Advances in diagnosis and treatment of Helicobacter pylori infection.

Helicobacter pylori is a Gram-negative motile bacterium causative agent of acute and chronic digestive and extra-digestive human infections. According to different reports worldwide, H. pylori symptomatic and asymptomatic infections are a global problem. The statistical investigations show a percentage of 50 for people who are involved in H. pylori acute/chronic digestive and/or extra-digestive infections around the world. This review focuses on digestive and extra-digestive diseases caused by H. pylori, the related virulence factors, diagnostic techniques including non-invasive and invasive diagnostics and treatment. There is an abundance of diagnostics for detection and identification of H. pylori. The availability, cost, and the condition of test performance may differ from place to place. To increase the level of reliability in association with diagnostic tools for detecting H. pylori, several techniques must be applied at once as multi-diagnostic technique. Furthermore, there are several pharmacotherapies which can be used for complete eradication of H. pylori infection.

Susceptibility patterns and cross-resistance of antibiotics against Pseudomonas aeruginosa isolated from burn patients in the South of Iran.

Pseudomonas aeruginosa plays a prominent role in serious infections in burn patients. Rapid acquisition of multi-drug resistance leads to high morbidity and mortality, especially in burn centers. Ten antibiotics, which were widely used in our burn patients were selected. MICs for imipenem, mropenem, cefepime, ceftazidime, cafoparazone/sulbactam, ticarcillin/clavulanate, piperacillin/tazobactam, ciprofloxacin, tobramycin and amikacin to 70 strains of P. aeruginosa, which were isolated from burn patients were determined by the E-test method (AB Biodisk, Sweden). Extended-spectrum beta-lactamase, group I inducible beta-lactamases and metallo-beta-lactamase activities were also determined. Imipenem and meropenum were the most active in vitro antibacterial agents followed by ciprofloxacin (p<0.05), whereas, ticarcillin/clavulanate was the least active. Almost all (98-100%) of the resistant isolates also showed cross-resistance to cefepime. The majority of imipenem and meropenem resistant isolates (85-100% and 76-100%) demonstrated cross-resistance to all the other antibiotics. ESBLs were detected in only three (4.3%) isolates, whereas, inducible beta-lactamase was observed in eight (11.4%) isolates. Metallo-beta-lactamase was detected in none of the isolates. Almost all of the antibiotic resistant isolates also showed cross-resistance to the majority of penicillins and cephalosporins with or without beta-lactamase inhibitors, from which ticarcillin/clavulanate demonstrated this phenomenon at the highest level.

Effect of cyclophosphamide on the course of Candida albicans infection in normal and vaccinated mice.

OBJECTIVE: To evaluate the immunomodulating effect of cyclophosphamide (Cy) on the course of Candida albicans (C. albicans). METHODS: We performed this study in the Shiraz Medical School, Shiraz, Iran during April to November 2003. Five groups of 10 mice (vaccinated group) were immunized by 5 equal injections of 2 x 10(5), 2.5 x 10(5) and 3 x 10(5) of the organism intraperitoneally. Then, the group received Cy on day zero and was challenged with lethal doses of C. albicans (7.74 x 10(5) colony forming unit) on days zero, one, 3, 6 and 12 post-Cy injection. Another 5 equal groups of 10 mice (non-vaccinated group) received Cy on day zero and similar to vaccinated ones were challenged with lethal doses of the organism too. The control groups received just Cy on day zero and were sacrificed on days zero, one, 3, 6 and 12 days post-Cy injection. We performed the hemogram and the spleen and studied the renal tissues microscopically and macroscopically. RESULTS: In vaccinated group, we observed an increase in survival time and in spleen and renal weights were visible while in non-vaccinated ones, a significant decrease was also observed on days one and 3 and an increased on days 6 and 12 post-Cy injection. We observed atrophy and necrosis in the spleen while inflammation and necrosis were also observed in the kidneys on days one and 3. We noticed a significant hyperplasia in the white pulp on days 6 and 12 post-Cy injection. CONCLUSION: We conclude that hyperplasia in the white pulp of spleen and the increase in peripheral polymorphonuclears due to selective effects of Cy could effectively protect the animal against C. albicans infection.

Immunodominant antigens of Helicobacter pylori strains isolated from patients with different gastroduodenal diseases.

OBJECTIVE: To detect the immunogenic proteins in Helicobacter pylori (H. pylori) strains isolated from patients with different gastric diseases. METHODS: We performed this study in the Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran, during July 2003 to September 2004. Total proteins of H. pylori strains isolated from the gastric biopsies of 3 groups of patients were separated by 1D-SDS-PAGE and then blotted with the sera of their respective hosts. RESULTS: In SDS-PAGE the members of each group showed high correlation according to similarity in their patterns, resulting in considering them in the same cluster. The patterns of immunoblots differed from that of Coomassie Brilliant Blue stained gels. The blotting method did not recognize some of the protein bands in the SDS-PAGE. Only the bands of 106 and 45 kDa from H. pylori strains isolated from patients with gastric cancer were significantly (p<0.05) recognized specifically with the sera of their respective patients, and the band of 13 kDa was recognized specifically (p<0.05) with the sera of nonulceric patients. With the exception of these bands, in the patterns of blotting of the sera from all patients no significant differences were observed. CONCLUSION: By using 1D blotting methods we could find 2 antigenic protein bands (106 and 45 kDa) for H. pylori strains isolated from cancerous patients, and one (13 kDa) for the strains isolated from nonulceric patients, which were specifically recognized with their respective host.

Genotyping of the Helicobacter pylori cagA Gene Isolated From Gastric Biopsies in Shiraz, Southern Iran: A PCR-RFLP and Sequence Analysis Approach.

BACKGROUND: Cytotoxin-associated gene A (cagA) is an important virulence factor in the pathogenesis of Helicobacter pylori. OBJECTIVES: The aim of this study was to genotype the H. pylori cagA gene isolated from antral biopsies of patients with stomach symptoms, using a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. PATIENTS AND METHODS: A total of 161 gastric biopsies were collected from patients with stomach symptoms. After isolation of H. pylori from the biopsy culture, the cagA gene was assessed using PCR. The PCR products were then digested by the HinfI restriction endonuclease enzyme. A sample of each genotype was also subjected to direct sequencing for further analysis. RESULTS: From 161 antral biopsies, 61 (37.9%) were positive for H. pylori in culture. Overall, 24 cagA-positives were detected in the isolates. RFLP indicated three different genotypes (I, II, and III) of cagA with a frequency of 62.5%, 25%, and 12.5% among the isolates, respectively. Genotypes I and II of cagA were predominant in patients who had gastritis. However, genotype III was found in three patients with duodenitis and duodenal ulcers. Alignment of the nucleotide sequences of the three isolated genotypes, with H. pylori 26695 as a reference strain, revealed 12 inserted nucleotides in genotype III. When the sequence of genotype III was aligned with 15 additional H. pylori strains available in GenBank, the same inserted nucleotides were detected in six of them. CONCLUSIONS: Using the PCR-RFLP method, three distinctive H. pylori cagA genotypes were detected in antral biopsies. Genotype I, which was predominant among the isolates, was significantly associated with gastritis. However, the data showed that cagA genotype III may play a role in duodenitis and duodenal ulcers in patients infected with H. pylori.

High efficacy of gemifloxacin-containing therapy in Helicobacter Pylori eradication: A pilot empirical second-line rescue therapy.

BACKGROUND: Helicobacter pylori (H pylori) is a common gastric pathogen which is associated with chronic gastritis, peptic ulcer, and gastric cancer. It has worldwide distribution with higher incidence in developing countries. Gemifloxacin is a fluoroquinolone antibiotic with documented in vitro activity against H pylori. Considering that there is no clinical data to verify gemifloxacin efficacy in H pylori eradication, this pilot clinical trial was designed. METHODS: This prospective pilot study was performed during February 2014 to February 2015. A regimen of gemifloxacin (320 mg single dose) plus twice daily doses of amoxicillin1g, bismuth 240 mg, and omeprazole 20 mg for 14 days were prescribed for H pylori infected patients in whom a first-line standard quadruple therapy (clarithromycin-amoxicillin-bismuth-omeprazole) had failed. To confirm H pylori eradication a 13C-urea breath test was performed 4 weeks after treatment.Compliance and incidence of adverse effects were evaluated by questionnaires. RESULTS: A total of 120 patients were enrolled consecutively; out of which 106 patients achieved H pylori eradication; per-protocol and intention-to-treat eradication rates were 91.4% (95% CI: 85.5-97.6) and 88.3% (95% CI: 75.4-92.4) respectively. Three patients (2.5%) failed to take at least 80% of the drugs and excluded from the final analysis. Adverse effects were reported in 42% of patients, most commonly including nausea (15%) and diarrhea (13.3%), which was intense in 1 patient and led to the discontinuation of treatment. In total, 96.7% (116/120) of the patients took the medications correctly. CONCLUSION: This study revealed that gemifloxacin-containing quadruple therapy provides high H pylori eradication rate (≥90% PP cure rate), and this agent can be included in the list of second-line H pylori therapeutic regimens.

Molecular Genotyping of Shigella sonnei Strains Isolated From Children With Bloody Diarrhea Using Pulsed Field Gel Electrophoresis on the Total Genome and PCR-RFLP of IpaH and IpaBCD Genes.

BACKGROUND: Identification, understanding of antibiotic sensitivity patterns and molecular characterization of genetic elements of Shigella species are important because of both epidemiological and clinical indications in developing countries. OBJECTIVES: The aim of this study was to analyze molecular epidemiology of Shigella isolates recovered from children with diarrhea in Shiraz (Southern Iran), using IpaH and IpaBCD PCR-restriction fragment length polymorphism (RFLP), and to determine pulsed field gel electrophoresis (PFGE) patterns of total DNA of the S. sonnei isolates to find the clonality among these strains. PATIENTS AND METHODS: A total of 82 clinical strains of Shigella spp., S. sonnei (n = 61), S. flexneri (n = 16), Shigella boydii (n = 3) and S. dysenteriae (n = 2) isolated from the stool samples of 719 patients, aged two months to 14 years, with positive occult blood (OB) test were characterized based on their IpaH and IpaBCD genes PCR-RFLP patterns. Genomic DNAs of S. sonnei strains were analyzed by PFGE. RESULTS: All Shigella isolates were positive for both invasive genes and showed homogeneous profiles for such genes except for two S. sonnei strains, which had IpaH bands with different sizes and PCR-RFLP profiles. Forty palsotypes were determined among the 41 S. sonnei strains. Sample patterns were divided into two groups based on the drawn dendrogram with a similarity range of 70% to 100%. CONCLUSIONS: The results revealed that the strains under study could be epidemically related. It seems that an alternative subtyping method is needed to study the relationship among clinical S. sonnei strains and their transmission. Here, we reported for the first time, two strains of S. sonnei with a different PCR-RFLP pattern for IpaH gene.

Characteristics of 26 kDa antigen of H. Pylori by Monoclonal Antibody.

Alkylhydroperoxide reductase (AhpC, the 26 kDa antigen) is one of the abundant antioxidant enzymes in Helicobacter pylori and seems to have a good potential for use in development of immunoassays to detect H. pylori infection in clinical specimens. This study aimed to investigate some properties of this antigen by the produced monoclonal antibodies. Five established hybridoma cell lines secreting monoclonal antibodies (MAbs) against 26 kDa antigen of H. pylori were cultivated and MAbs were purified by affinity chromatography. Subsequently, MAbs were conjugated with biotin, and different combinations of capture and tracer antibodies used in sandwich ELISA. Immunoblotting of bacterial extracts were performed to estimate aggregation status of the antigen. Release of antigen from the cultivated bacteria on solid media was examined by sandwich ELISA, and also, existence of interference in fecal extract was investigated by immunoblotting and sandwich ELISA. Our findings showed that the MAbs against 26 kDa antigen of H. pylori could recognize three bands of nearly 25 kDa, 50 kDa, and 75 kDa in immunoblotting. This study also indicated presence of more antigens in the culture medium around the bacteria than the bacterial extract itself. The results of sandwich ELISA and immunoblotting on fecal extracts suggest the presence of interfering agents that prevent detection of antigen by antibody in ELISA but not in immunoblotting. In this study the oligomerization of the 26 kDa antigen, presence of interfering agents in stool matrix, and release of antigen to outside of bacteria, were demonstrated.