Prevalence of Helicobacter pylori in symptomatic patients and detection of clarithromycin resistance using melting curve analysis

AbstractBackground:Clarithromycin is often a component of combination therapies for Helicobacter pylori eradication; however, increases in resistance rates have decreased the success of the treatment.Objective:This study was designed to determine the prevalence of H pylori infection in symptomatic patients and to detect clarithromycin resistance rates using melting curve analysis.Methods:Patients scheduled for upper endoscopy at the Endoscopy Unit of the Department of Gastroenterology, Duzce University, Medical Faculty Hospital, Konuralp/Duzce, Turkey, were assessed for enrollment in the study. Two pairs of gastric biopsy specimens (antrum and corpus) were obtained from each study patient. Histopathologic examination, rapid urease test, culture, and polymerase chain reaction (PCR) of the specimens were used to identify H pylori infection. Clarithromycin resistance was detected using melting curve analysis.Results:Seventy-five patients (41 women, 34 men; mean [SD]age, 42.6 [14.5] years [range, 17–70 years]) were included in the study. Using histopathology and rapid urease test, H pylori was detected in 40 (53.3%) of the 75 specimens. H pylori was detected using PCR in 40 (53.3%) specimens and by culture in 10 (13.3%) specimens. The specificity and sensitivity of PCR and culture were interpreted by comparing them with the results of histopathologic examination and urease tests. The specificity and sensitivity of PCR were 68.6% and 72.5%, respectively, and the specificity and sensitivity of culture were 97.1% and 22.5%, respectively. Of the 40 isolates, 21 (52.5%) were susceptible to clarithromycin, 12 (30.0%) were resistant, and a mixed susceptibility pattern was detected in 7 (17.5%) specimens. H pylori isolates from 19 (79.2%) of the 24 patients who had formerly used clarithromycin showed clarithromycin resistance.Conclusions:The prevalence of H pylori infection was 53.3% for the symptomatic patients in this study, and 47.5% of the isolates showed clarithromycin resistance using melting curve analysis. The PCR-based system used in this study was accurate for the detection of H pylori infection as well as clarithromycin susceptibility testing directly in biopsy specimens

The Helicobacter pylori Genome Project : insights into H. pylori population structure from analysis of a worldwide collection of complete genomes

Helicobacter pylori, a dominant member of the gastric microbiota, shares co-evolutionary history with humans. This has led to the development of genetically distinct H. pylori subpopulations associated with the geographic origin of the host and with differential gastric disease risk. Here, we provide insights into H. pylori population structure as a part of the Helicobacter pylori Genome Project (HpGP), a multi-disciplinary initiative aimed at elucidating H. pylori pathogenesis and identifying new therapeutic targets. We collected 1011 well-characterized clinical strains from 50 countries and generated high-quality genome sequences. We analysed core genome diversity and population structure of the HpGP dataset and 255 worldwide reference genomes to outline the ancestral contribution to Eurasian, African, and American populations. We found evidence of substantial contribution of population hpNorthAsia and subpopulation hspUral in Northern European H. pylori. The genomes of H. pylori isolated from northern and southern Indigenous Americans differed in that bacteria isolated in northern Indigenous communities were more similar to North Asian H. pylori while the southern had higher relatedness to hpEastAsia. Notably, we also found a highly clonal yet geographically dispersed North American subpopulation, which is negative for the cag pathogenicity island, and present in 7% of sequenced US genomes. We expect the HpGP dataset and the corresponding strains to become a major asset for H. pylori genomics

Persistence of Burkholderia pseudomallei, B. multivorans and B. cenocepacia : differential analysis by proteomics and activation of phosphoinositide-3 kinase

Persistent infection in mammalian hosts is one intriguing aspect of the study of human diseases. In order to gain insight into the establishment of persistence, this thesis will evaluate the bacterial and host components associated with persistence of two groups within the Burkholderia genus: Burkholderia pseudomallei and Burkholderia cepacia complex. Burkholderia pseudomallei causes septicemic melioidosis with a high rate of relapse; however microbial determinants of relapse are unknown. Proteins were analyzed from sequential B. pseudomallei isolates from primary and relapsing melioidosis. Analysis by iTRAQ revealed that factors required for nitric oxide detoxification (HmpA) and necessary for anaerobic growth (ArcA, ArcC, and ArcB) were highly expressed in the relapse isolate. 2D-PAGE revealed up-regulation of a putative hcp-1 protein in the primary isolate, and flagellin and HSP20/alpha crystalline in the relapse isolate. These observations provide targets for further analysis of latency and virulence of B. pseudomallei in patients with relapsing melioidosis. The role of the host phosphoinositide3-kinase/Akt (PI3K/Akt) signaling pathway in the interaction of Burkholderia cepacia complex (Bcc) species with human immortalized macrophages was explored. B. cenocepacia and B. multivorans are the most clinically important and prevalent of the Bcc members and are serious opportunistic pathogens in cystic fibrosis (CF) patients. This study evaluated whether these pathogens can differentially induce PI3K/Akt signaling pathway, which is important in cell survival. B. cenocepacia activated PI3K/Akt in multiple cell types and this activation proceeded faster after exposure to B. multivorans than to B. cenocepacia in macrophages. Both species promoted cell survival by preventing caspase 9 cleavage, and bacteria internalization was necessary for this process. Unlike, B. cenocepacia, B. multivorans induced PI3K/Akt mediated anti-apoptotic effect, faster NF-κB activation, and IκBα phosphorylation that was PIK3/Akt dependent. Macrophages exposed to B. cenocepacia induced greater proinflammatory cytokines release compared to B. multivorans. Internalization into macrophages of both species was PI3K regulated. These findings provide novel insights into the pathogenic mechanism underlying Bcc infection in CF. Together; this thesis provides data for the identification of mechanisms leading to the persistence of these bacteria in melioidosis and CF and may inform the design of therapeutic approaches for treatment in these diseases.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat

Quick versus Quantitative: Evaluation of Two Commercial Real-Time PCR Assays for the Detection of Pneumocystis jirovecii from Bronchoalveolar Lavage Fluids

ABSTRACT Two commercial real-time PCR assays for the detection of Pneumocystis jirovecii were compared, the quantitative RealStar P. jirovecii assay and the qualitative DiaSorin P. jirovecii assay, the latter of which can be used without nucleic acid extraction. Archived bronchoalveolar lavage (BAL) specimens (n = 66), previously tested by molecular methods, were tested by both assays, and the results were compared to the respective original result. The RealStar P. jirovecii assay demonstrated good positive percent agreement (PPA) (90% [95% confidence interval (CI), 72 to 97%]; 27/30) and negative percent agreement (NPA) (100% [95% CI, 88 to 100%]; 36/36) with the reference method. The DiaSorin P. jirovecii assay concordantly detected P. jirovecii in 19 of 24 positive BAL samples (PPA = 73% [95% CI, 52 to 88%]). All negative BAL samples gave concordant results (NPA = 100% [95% CI, 87 to 100%]; 34/34). Discordant results occurred mostly in samples with low fungal loads. In conclusion, the RealStar assay demonstrated good concordance with reference results, and the DiaSorin P. jirovecii assay performed well for negative BAL and positive BAL samples with P. jirovecii concentrations of greater than 260 copies/mL. IMPORTANCE Pneumonia, caused by the opportunistic fungus Pneumocystis jirovecii, poses a significant risk for immunocompromised individuals. Laboratory testing for P. jirovecii is progressively shifting toward the use of molecular tests such as real-time PCR; however, this is often performed at reference laboratories. Many frontline laboratories are looking into improving their service and reducing turnaround times for obtaining P. jirovecii results by bringing molecular P. jirovecii testing in-house. We evaluated and compared two commercial real-time PCR assays with different workflows for the detection of P. jirovecii from bronchoalveolar lavage specimens. The RealStar P. jirovecii assay requires nucleic acid extraction and provides a quantification of fungal load for positive samples. The DiaSorin P. jirovecii assay offers a simple workflow without nucleic extraction from patient samples and qualitative results. Results from this study provide valuable information on performance and workflow considerations for laboratories that wish to implement P. jirovecii molecular testing

Use of copper formulations in hospitals

Objective: To evaluate three formulations of copper (Cu) based self-sanitizing surfaces for antimicrobial efficacy and durability over one year in inpatient clinical areas and laboratories. Design: Randomized control trial. Setting: Three Cu formulations were assessed a) solid alloy 80% Cu 20% Ni (integral copper), b) spray–on 80% Cu 20% Ni (spray-on) and c) 16% composite Cu-impregnated surface. Coupons (1cm2 ) of the three products and control surgical grade (AISI 316) stainless steel (SS) were inserted into gaskets and adhered onto clinical carts used in patient care areas (including Emergency and Maternity units) (n=480) and on microbiology laboratory bench workspaces (n=240). The microbial burden and assessment of resistance to wear, corrosion, and material compatibility were determined every three months. Three tertiary care Canadian adult and one paediatric/maternity hospital participated. Results: Cu formulations used on inpatient units statistically significantly reduced bacterial bioburden compared to SS at months 3 and 6. Only the integral product had significantly less bacteria compared to SS at month 12. There were no statistically significant differences in microbial burden between Cu formulations and SS coupons on microbiology laboratory benches where bacterial counts were low overall. All mass changes and corrosion rates of the formulations were acceptable by engineering standards. Conclusions: Cu surfaces vary in their antimicrobial efficacy after one year in-hospital use. Frequency of cleaning and disinfection influences the impact of copper with the greatest reduction in microbial bioburden seen in clinical areas compared to the microbiology laboratory where cleaning/disinfection occurred multiple times daily.Applied Science, Faculty ofMedicine, Faculty ofNon UBCMaterials Engineering, Department ofPathology and Laboratory Medicine, Department ofReviewedFacultyResearcherPostdoctoralGraduat

Helicobacter pylori from Peruvian amerindians : traces of human migrations in strains from remote Amazon, and genome sequence of an Amerind strain

BACKGROUND: The gastric pathogen Helicobacter pylori is extraordinary in its genetic diversity, the differences between strains from well-separated human populations, and the range of diseases that infection promotes. PRINCIPAL FINDINGS: Housekeeping gene sequences from H. pylori from residents of an Amerindian village in the Peruvian Amazon, Shimaa, were related to, but not intermingled with, those from Asia. This suggests descent of Shimaa strains from H. pylori that had infected the people who migrated from Asia into The Americas some 15,000+ years ago. In contrast, European type sequences predominated in strains from Amerindian Lima shantytown residents, but with some 12% Amerindian or East Asian-like admixture, which indicates displacement of ancestral purely Amerindian strains by those of hybrid or European ancestry. The genome of one Shimaa village strain, Shi470, was sequenced completely. Its SNP pattern was more Asian- than European-like genome-wide, indicating a purely Amerind ancestry. Among its unusual features were two cagA virulence genes, each distinct from those known from elsewhere; and a novel allele of gene hp0519, whose encoded protein is postulated to interact with host tissue. More generally, however, the Shi470 genome is similar in gene content and organization to those of strains from industrialized countries. CONCLUSIONS: Our data indicate that Shimaa village H. pylori descend from Asian strains brought to The Americas many millennia ago; and that Amerind strains are less fit than, and were substantially displaced by, hybrid or European strains in less isolated communities. Genome comparisons of H. pylori from Amerindian and other communities should help elucidate evolutionary forces that have shaped pathogen populations in The Americas and worldwide