Comparative Evaluation of Five Culture Media with Triplex PCR Assay for Detection of Methicillin-Resistant Staphylococcus aureus

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) is responsible for nosocomial and community-acquired infections. Hence, rapid and accurate laboratory diagnosis of MRSA is a vital constituent of control measures. The present study evaluated five different methods for the identification of MRSA. A total of 207 S. aureus clinical isolates that consisted of 89 MRSA and 118 methicillin-susceptible S. aureus (MSSA) strains confirmed by PCR were tested. MRSA strains were evaluated by five different methods: chromogenic MRSA agar (CMRSA), oxacillin resistance screening agar base (ORSAB), mannitol salt oxacillin agar (MSO), mannitol salt cefoxitin agar with two different concentrations of cefoxitin [4 μg/ml (MSC-4) and 6 μg/ml (MSC-6)]. The results of the different methods were compared to mecA PCR as the gold standard. MSC-6 showed only six false-positive MRSA in comparison with PCR. The sensitivities and specificities of MSC-6, MSC-4, MSO-4, ORSAB, and CMRSA were as follows: 98.9/94.9%, 100/83.1%, 89.9/87.3%, 97.8/96.6%, and 95.5/94.9%, respectively. In comparison with PCR, it was found that both MSC-6 and ORSAB were relatively the least expensive screening tests (0.70 and0.70 and 1.00, respectively). In conclusion, all methods were comparable, but MSC-6 was the least expensive medium for MRSA screening.     

Is Antrum or Corpus the Best Site for Culture of Helicobacter pylori?

Background Isolating Helicobacter pylori on culture media and performing antibiotic susceptibility testing is potentially the most useful tool for guiding antibiotic therapy, especially when antimicrobial resistance is suspected. The aim of this study was to determine whether the yield of H. pylori culture was related to the site from which the gastric specimen was obtained either before or after therapy.
Methods. Gastric mucosal biopsies from the antrum and the corpus of the stomach were cultured. H. pylori status was determined by histological assessment using the Genta stain.
Results. Fifty-two patients with documented H. pylori infection were studied: Twenty-three were tested before antibiotic therapy and 29 after therapy had failed. In 47 patients (90%), both antral and corpus culture specimens were positive. In 5 patients (10%), only one site was positive, with three false-negative antral and two false negative corpus cultures. The overall sensitivity of culture in detecting H. pylori infection was 95% (95% confidence interval = 89–98%) and was not significantly different for the antrum or corpus, either before or after therapy.
Conclusion. Culture of gastric biopsies from either the antrum or the corpus has an excellent diagnostic yield even in patients who failed antimicrobial therapy.     

Performance of individual Helicobacter pylori antigens in the immunoblot-based detection of H. pylori infection

To develop a specific line blot (LB) for supporting ELISA-based serodiagnosis of Helicobacter pylori infection, individual native/recombinant H. pylori antigens were evaluated with respect to their reactivity with both serum IgG and IgA from 156 dyspeptic screening patients (67% H. pylori positive). Of 13 antigens, HP0175, p17, and p19 revealed highest positive likelihood ratios for H. pylori-specific IgG (> 5.0) and were selected as LB substrates, in addition to the established virulence markers VacA and CagA. For validation, the LB was compared to a commercial whole-cell-lysate-based ELISA by parallel (re-)analysis of 156 screening sera, 22 sera from diabetes mellitus patients and 15 sera from follow-up patients after H. pylori eradication. In screening patients, the combined use of IgG ELISA and LB revealed a sensitivity, specificity, and accuracy of 94%, 81%, and 90%, respectively, whereas IgG ELISA alone exhibited a low specificity of 75%. In diabetic and follow-up patients, IgA ELISA exhibited high accuracy of 89% and 93%, respectively, whereas IgG detection was unreliable (accuracy < 80%). In conclusion, using HP0175, p17, p19, CagA, and VacA as LB substrates significantly improves the specificity of anti-H. pylori IgG analysis, providing a reliable tool for (1) confirmation/refutation of ELISA-based screening results and (2) assessment of the CagA/VacA status.     

Is there seasonal periodicity in the prevalence of Helicobacter pylori?

The objective of the present study was to evaluate seasonal periodicity in the prevalence of Helicobacter pylori. A prospective study was performed on 1076 consecutive patients who were investigated in our hospital over a 3-year span because of epigastric complaints. Our findings indicate a significant accumulation of positive Helicobacter pylori tests in October. Gastric acidity, gender, and age did not influence Helicobacter pylori infection significantly. There was no significant correlation between potential seasonal influence on the diagnosis of ulcer disease and the seasonal fluctuation of Helicobacter pylori infection. The seasonality was confirmed by cosinor analysis for the absolute frequencies of H. pylori infections and also for the number of cases positive for H. pylori per number of presenting patients per month. A seasonal concept of a sensitivity threshold for positive Helicobacter pylori testing is introduced, taking into account such factors as immune system, nutrition, and medication status.     

Different Expression of Helicobacter pylori Gastritis in Children: Evidence for a Specific Pediatric Disease?

Background Infection with Helicobacter pylori causes active chronic gastritis. Once the infection is acquired, gastritis will persist for almost the rest of one's life. To date, very few data are available on H. pylori gastritis in relation to age. Therefore, we attempted to inestigate whether H. pylori gastritis in children exhibits features different from H. pylori gastritis in adults of two different age groups. Materials and Methods. Fifty consecutive children with a median age of 11 years (range, 3–18 years) were compared with two groups of 50 adult patients, one group with a median age of 43 (range, 19–56 years) and another group with a median age of 70 years (range, 59–86 years). All patients had H. pylori gastritis unrelated to active peptic ulcer disease. Two biopsy specimens were taken from the antrum and two from the corpus, and the following gastritis parameters were evaluated: degree and activity of gastritis, H. pylori colonization, replacement of foveolar epithelium by regenerative epithelium, mucous depletion, presence of atrophic gastritis with intestinal metaplasia, and presence of lymphoid follicles. Results. Degree and activity of gastritis, extent of H. pylori colonization, degree of replacement by regenerative epithelium, extent of mucous depletion, degree of atrophic gastritis with intestinal metaplasia, and the presence of lymphoid follicles in the antrum, as well as the presence of lymphoid follicles in the corpus differed significantly (chi-square test: p < .05). All these differences—except the once frequent occurrence of atrophic gastritis with intestinal metaplasia in adults—were attributable to a higher expression of these gastritis parameters in children. Conclusions. We conclude that H. pylori gastritis, particularly in the antrum, is more severely expressed in childhood. One reason for this might be a child-specific immune response to an infection with H. pylori. Alternatively, infection may represent a pediatric disease characterized by a nonatrophic, highly expressed form of gastritis, which changes its appearance once the host becomes adapted over time.     

Lipid peroxidation in chronic gastritis; any influence of Helicobacter pylori?

In an attempt to investigate the significance of lipid peroxidation in the pathogenesis of gastritis associated with or without Helicobacter pylori infection, malonodialdehyde (MDA) levels were measured by the thiobarbiturate assay in the gastric juice of 101 patients undergoing upper GI endoscopy and correlated with histopathological findings. Elevated MDA levels were found in all patients with gastritis compared with controls. MDA levels were significantly correlated with the extent of the mucosal inflammation and with disease activity in patients with reactive gastritis. In patients with H. pylori associated gastritis MDA levels were not correlated with disease activity but rather with the degree of atrophy. In this case, MDA levels were equal or even less than in patients with reactive gastritis. MDA levels were not affected by the history of consumption of PPIs, of H(2)-blockers or of NSAIDs over the last month before the endoscopy. It is concluded that lipid peroxidation is a mechanism involved in the pathogenesis of gastritis associated or not to H. pylori infection.     

Is Helicobacter pylori the cause of dyspepsia?

OBJECTIVE--To determine the association between infection with Helicobacter pylori and dyspepsia. DESIGN--Cross sectional study of dyspeptic subjects and age and sex matched controls identified by a questionnaire survey of all inhabitants aged 20-69. (Endoscopy, histological examination, and microbiological examinations of biopsies from the gastric mucosa were performed blind.) SETTING--Population based survey in Sørreisa, Norway. SUBJECTS--All 782 dyspeptic subjects (excluding those with a previous history of peptic ulcer, gall stones or kidney stones, and coronary heart disease) and controls were offered an endoscopy, of whom 309 dyspeptic subjects and 310 controls attended. MAIN OUTCOME MEASURES--Prevalences of endoscopic and histological diagnoses and of cultures positive for H pylori. RESULTS--A high prevalence of positive cultures, increasing with age, was found in both dyspeptic subjects (48%) and non-dyspeptic controls (36%) (p = 0.004). Positive cultures in both dyspeptic subjects and controls were strongly associated with histological gastritis (70%, 95% confidence interval 65.5 to 85.3; 60%, 52.7 to 67.7, respectively) and peptic ulcer (92%, 61.5 to 99.8; 64.1, 9.4 to 99.2, respectively). Only 3% of subjects with a histologically non-inflamed gastric mucosa had this infection (dyspeptic subjects 2%, 0.2 to 7.0; controls 4%; 1.2 to 8.8). CONCLUSIONS--The relation between dyspeptic symptoms and H pylori is dubious; H pylori seems to have a pathogenetic role in gastritis and may be a contributing factor but not a cause of peptic ulcer.     

Structural Basis for the Inhibition of Helicobacter pylori α-Carbonic Anhydrase by Sulfonamides

Periplasmic α-carbonic anhydrase of Helicobacter pylori (HpαCA), an oncogenic bacterium in the human stomach, is essential for its acclimation to low pH. It catalyses the conversion of carbon dioxide to bicarbonate using Zn(II) as the cofactor. In H. pylori, Neisseria spp., Brucella suis and Streptococcus pneumoniae this enzyme is the target for sulfonamide antibacterial agents. We present structural analysis correlated with inhibition data, on the complexes of HpαCA with two pharmacological inhibitors of human carbonic anhydrases, acetazolamide and methazolamide. This analysis reveals that two sulfonamide oxygen atoms of the inhibitors are positioned proximal to the putative location of the oxygens of the CO₂ substrate in the Michaelis complex, whilst the zinc-coordinating sulfonamide nitrogen occupies the position of the catalytic water molecule. The structures are consistent with acetazolamide acting as site-directed, nanomolar inhibitors of the enzyme by mimicking its reaction transition state. Additionally, inhibitor binding provides insights into the channel for substrate entry and product exit. This analysis has implications for the structure-based design of inhibitors of bacterial carbonic anhydrases.     

Helicobacter pylori pediatric infection changes FcεRI expression in dendritic cells and Treg profile in vivo and in vitro

H. pylori infection shows an inverse relationship with allergies. Dendritic cells regulate mucosal immune responses including the induction of T regulatory cells which are fundamental in Helicobacter pylori-induced dampening of allergies. In this respect expression of high-affinity IgE receptor (FcεRI) has been associated with a regulatory dendritic cell profile. Therefore we aimed to evaluate possible mechanisms by which H. pylori infection might modify atopy in pediatric patients. Here we show that H. pylori-infected children exhibited both increased expression of FcεRI on peripheral myeloid and plasmacytoid dendritic cells and higher levels of Foxp3 and Latency Associated Peptide on T regulatory cells. Moreover, exposure to H. pylori drove increased FcεRI expression and IL-10 secretion by both pediatric H. pylori-exposed monocyte derived dendritic cells and T cells. Finally, we show a positive correlation between expression of FcεRI in circulating myeloid DCs and total Treg cells, suggesting that in children, H. pylori infection may have a modulating role in atopy, mediated by both altered surface expression of FcεRI on children's DC and an increased T regulatory cell profile.     

Systematic Comparative Evaluation of Methods for Investigating the TCRβ Repertoire

High-throughput sequencing has recently been applied to profile the high diversity of antibodyome/B cell receptors (BCRs) and T cell receptors (TCRs) among immune cells. To date, Multiplex PCR (MPCR) and 5’RACE are predominately used to enrich rearranged BCRs and TCRs. Both approaches have advantages and disadvantages; however, a systematic evaluation and direct comparison of them would benefit researchers in the selection of the most suitable method. In this study, we used both pooled control plasmids and spiked-in cells to benchmark the MPCR bias. RNA from three healthy donors was subsequently processed with the two methods to perform a comparative evaluation of the TCR β chain sequences. Both approaches demonstrated high reproducibility (R² = 0.9958 and 0.9878, respectively). No differences in gene usage were identified for most V/J genes (>60%), and an average of 52.03% of the CDR3 amino acid sequences overlapped. MPCR exhibited a certain degree of bias, in which the usage of several genes deviated from 5’RACE, and some V-J pairings were lost. In contrast, there was a smaller rate of effective data from 5’RACE (11.25% less compared with MPCR). Nevertheless, the methodological variability was smaller compared with the biological variability. Through direct comparison, these findings provide novel insights into the two experimental methods, which will prove to be valuable in immune repertoire research and its interpretation.     

Evaluation of Iodide and Iodate for Adsorption–Desorption Characteristics and Bioavailability in Three Types of Soil

Adsorption–desorption of iodine in two forms, viz., iodide (I⁻) and iodate (IO₃⁻), in three types of soil were investigated. The soils were: red soil developed on Quaternary red earths (REQ)— clayey, kaolintic thermic plinthite Aquult, Inceptisol soil (IS) and alluvial soil (AS)—Fluvio-marine yellow loamy soil. The isothermal curves of iodine adsorption on soils were described by Langmuir and Freundlich equation, and the maximum adsorption values (y _m) were obtained from the simple Langmuir model. As compared with the iodide, the iodate was adsorbed in higher amounts by the soils tested. Among three soils, the REQ soil adsorbed more iodine (I⁻ and IO₃⁻) than the IS and AS. The distribution coefficient (K _d) of iodine in the soils decreased exponentially with increasing iodine loading concentration. Desorption of iodine in soil was increased correspondingly with increasing adsorption values. The REQ soil had a greater affinity for iodine than the IS and AS at the same iodine loadings. In the pot experiment cultivated with pakchoi (Brassica chinensis L.) and added with two exogenous iodine sources, the iodide form was quickly taken up by pakchoi and caused more toxicity to the vegetable. The rate of iodine loss from soil was higher for iodide form as compared with the iodate. The iodine bioavailability was the highest but the persistence was the weakest in AS among the three soils tested, and the REQ soil showed just the opposite trend to that of the AS soil. This study is of theoretical importance to understand the relationship between iodine adsorption–desorption characteristics and their bioavailability in different soils and it also has practical implications for seeking effective alternatives of iodine biofortification to prevent iodine deficiency disorders.     

Cholesterol glucosylation–based survival strategy in Helicobacter pylori

Helicobacter pylori is a major chronic health problem, infecting more than half of the population worldwide. H. pylori infection is linked with various clinical complications ranging from gastritis to gastric cancer. The resolution of gastritis and peptic ulcer appears to be linked with the eradication of H. pylori. However, resistance to antibiotics and eradication failure rates are reaching alarmingly high levels. This calls for urgent action in finding alternate methods for H. pylori eradication. Here, we discuss the recently identified mechanism of H. pylori known as cholesterol glucosylation, mediated by the enzyme cholesterol-α-glucosyltransferase, encoded by the gene cgt. Cholesterol glucosylation serves several functions that include promoting immune evasion, enhancing antibiotic resistance, maintaining the native helical morphology, and supporting functions of prominent virulence factors such as CagA and VacA. Consequently, strategies aiming at inhibition of the cholesterol glucosylation process have the potential to attenuate the potency of H. pylori infection and abrogate H. pylori immune evasion capabilities. Knockout of H. pylori cgt results in unsuccessful colonization and elimination by the host immune responses. Moreover, blocking cholesterol glucosylation can reverse antibiotic susceptibility in H. pylori. In this work, we review the main roles of cholesterol glucosylation in H. pylori and evaluate whether this mechanism can be targeted for the development of alternate methods for eradication of H. pylori infection.     

Statistical Methods for Integrating Randomized Clinical Studies for Evaluation of Combination Treatment Therapy

In regulatory applications, evaluation of a combination drug at a fixed dose is based on pairwise comparisons of the combination with its component drugs. These comparisons generate the least expected gain of the combination relative to its components as the key effect parameter for evaluation. Two test methods are developed for the evaluation to combine multiple clinical studies that are deemed combinable. One test method is based on a linear combination of the Min tests from individual studies. In the other test method, weighted estimators are first derived for the pairwise comparisons between the combination and the respective components by combining the studies. A Min test is then applied to these estimators. The latter test method tends to be more powerful than the former test method. A test-based confidence interval is constructed for the least expected gain of the combination relative to its components. A test for heterogeneity across studies is also developed.