Grażyna Gościniak

Helicobacter pylori antigens in stool specimens of gastritis children before and after treatment.

Background Various testing methods are successfully applied to the diagnosis of Helicobacter pylori infection, but noninvasive techniques are still needed for therapeutic monitoring, especially in children. In the search for new noninvasive techniques for the diagnosis of H. pylori infection, the authors evaluated an enzyme immunoassay for the detection of H. pylori antigen in stool (HpSA). Methods The authors studied 62 H. pylori–positive children with chronic gastritis and 45 control subjects. H. pylori infection was diagnosed using cultures and histology of gastric biopsy specimens and a stool antigen test before treatment (clarithromycin, amoxicillin, omeprazole for 7 days) and 4 weeks to 6 weeks after treatment. Results Before therapy, antigen in stool was detected in 55 of 62 H. pylori–positive patients, which indicates that the sensitivity of the HpSA test was 88.7%. Of the 45 control subjects (with negative culture and histology results), 43 had negative results for H. pylori in the stool test (specificity, 95.5%). After completion of therapy, eradication was obtained (and confirmed by culture and histology) in 53 of the 62 H. pylori–positive children (85.5%). Four weeks to 6 weeks after eradication therapy, the sensitivity, specificity, positive predictive value, and negative predictive value of the stool antigen (HpSA) test were 88.9%, 96.2%, 80%, and 98%, respectively. Conclusions The accuracy of the HpSA test for the detection of H. pylori in human stool 4 weeks to 6 weeks after treatment is comparable with the accuracy of the culture results. The stool antigen (HpSA) test was found to be a useful method for posttreatment eradication testing of H. pylori infection in children.

Seroprevalence of Helicobacter pylori infection in Polish children and adults depending on socioeconomic status and living conditions

PURPOSE Helicobacter pylori (H. pylori) is one of the causes of gastritis, peptic ulcer disease, gastric cancer and MALT-lymphoma. The frequency of H. pylori infection is different in various regions of the world and dependent on age, socioeconomic and hygiene status. The objective of this study was to assess seroprevalence and the associated socioeconomic and sociodemographic characteristics influencing H. pylori infection in children and adults in Polish population. MATERIAL/METHODS In multicenter epidemiological studies, H. pylori infection occurrence was assessed in Poland in the years 2002 and 2003. The seroprevalence of H. pylori infection diagnosis was based on IgG anti-H. pylori antibodies concentration above 24 UI/ml, which was measured using ELISA test. The study included 6565 subjects: 3307 adults (50.37%) and 3258 children (49.63%). RESULTS Positive result was observed in 3827 subjects (58.29%), i.e. 1043 children (32.01%) and 2784 adults (84.19%). H. pylori infection prevalence was greater in children of poor economic status, who were born in a rural area, lived in crowded houses with no running tap water and with toilet outside the house, and who did not observe hygiene rules. In adults, the factors predisposing to higher probability of being H. pylori infected included: being born in a rural area, having low family income and elementary education, smoking tobacco, drinking high proof alcohols as well as not observing of hygiene rules. CONCLUSIONS Improvement of socioeconomic status, sanitary and hygienic conditions and the education of the society might decrease H. pylori infection prevalence in children and in adults.

Antimicrobial susceptibility of Helicobacter pylori isolates from Lower Silesia, Poland.

Introduction In recent years the failure of standard therapy for Helicobacter pylori infections has been observed, which results primarily from the increasing resistance of H. pylori strains to antibiotics. The aim of the study was to estimate the prevalence of antimicrobial resistance of H. pylori strains isolated from adult symptomatic patients with primary infection in the Lower Silesia Region in Poland. Material and methods One hundred and seventy-eight adults aged 19–89 years with dyspeptic symptoms suggesting gastroduodenal pathology were enrolled in the study. The study was performed in the years 2008–2011. Fifty H. pylori strains were isolated from gastric biopsy samples of examined patients. Antimicrobial susceptibility to 6 drugs (amoxicillin (AM), clarithromycin (CH), metronidazole (MZ), tetracycline (TC), levofloxacin (LEV), and rifabutin (RB)) was tested by the gradient-diffusion method (E-test method). Results The incidence of H. pylori infection among examined patients was 35%. From 50 isolated H. pylori strains, 24% showed resistance to CH, 42% to MZ and 8% to LEV alone. Multidrug resistance was detected in 26% of strains, whereas 20% of isolates were resistant to MZ and CH. Examined strains were fully susceptible to AM, TC and RB. Conclusions Resistance to clarithromycin strains isolated from adults of the Lower Silesia Region in Poland is high and is almost always associated with resistance to metronidazole (CH + MZ). It is necessary to continuously monitor H. pylori resistance to drugs used in therapy, especially to clarithromycin. Verification of the existing recommendations of eradication therapy is also needed.

Prevalence of Helicobacter pylori cagA, vacA, iceA, babA2 genotypes in Polish children and adolescents with gastroduodenal disease.

INTRODUCTION Infection with Helicobacter pylori is a major cause of chronic gastritis and peptic ulcer disease in children and its consequences in adulthood can lead to serious complications, including in particular the development of gastric cancer. Our aim was to analyze the relationship between the occurrence of selected genes such as cagA, vacA, iceA, and babA2 determining pathogenicity of H. pylori strains and clinical outcome in children. MATERIAL AND METHODS The study was performed on H. pylori strains isolated from biopsies taken from 130 children and adolescents with non-ulcer dyspepsia (NUD), gastric and duodenal ulcers (PUD) and gastroesophageal reflux disease (GERD). Genes such as cagA, vacA (allelic variants: s1/ s2, m1/m2), iceA (allelic variants: iceA1, iceA2) and babA2 were determined by polymerase chain reaction (PCR). RESULTS The cagA gene was detected in 79/130 (60.8%) H. pylori isolates. The presence of the cagA gene was significantly associated with duodenal ulcer (p<0.05). The vacAs1/m1 genotype as more frequent in children with ulcers than in other groups, whereas the vacAs2/m2 genotype was more frequent in patients with gastritis and GERD. The iceA1, iceA2 and babA2 genes were present in 59/130 (45.4%), 27/130 (21%) and 30/130 (23.1%) of the strains, respectively. The vacAs1/cagA+ genotype was most frequently observed in strains isolated from children with PUD. The predominant genotype in children with NUD and GERD was vacAs2/cagA-/iceA1+/babA2-. CONCLUSION The study showed a high incidence of strains with increased virulence, possessing cagA, vacAs1 and iceA1 genes in symptomatic children with H. pylori infection.

Neutralisation of cytotoxic vacuolating activityby serum antibodies of Helicobacter pylori-infected patients

The study involved 196 H. pylori strains and 196 serum samples taken from the same patients. H. pylori strains were investigated for the production of vacuolating cytotoxin. Antibodies to the vacuolating cytotoxin produced by H. pylori were detected in the sera samples by neutralisation assay (on Intestine 407 cells) and ELISA. Of the 196 H. pylori strains tested, 80 (40.8%) were found to express vacuolating cytotoxic activity. The titres of cytotoxic nonconcentrated broth culture filtrates ranged from 1:2 to 1:128. The vacuolating assay was positive in 37.1% strains isolated from children, and in 50% strains isolated from adults. Cytotoxin-positive H. pylori strains were found more frequently in duodenal ulcer (71%) than in chronic gastritis (35.2%) patients, and this difference was statistically significant p < 0.05. Neutralising antibodies to vacuolating cytotoxin were present in 51% and 49% of the serum samples tested by neutralisation and ELISA, respectively. Duodenal ulcer patients had antibodies to vacuolating cytotoxin more frequently (p < 0.05) than chronic gastritis patients. Antibodies to cytotoxin were detected in the serum samples from patients infected by cytotoxic (100%) and noncytotoxic (18%) H. pylori strains.

Aminophosphinates against Helicobacter pylori ureolysis—Biochemical and whole-cell inhibition characteristics

Urease is an important virulence factor from Helicobacter pylori that enables bacterial colonization of human gastric mucosa. Specific inhibition of urease activity can be regarded as a promising adjuvant strategy for eradication of this pathogen. A group of organophosphorus inhibitors of urease, namely, aminophosphinic acid and aminophosphonic acid derivatives, were evaluated in vitro against H. pylori urease. The kinetic characteristics of recombinant enzyme activity demonstrated a competitive reversible mode of inhibition with Ki values ranging from 0.294 to 878 μM. N-n-Hexylaminomethyl-P-aminomethylphosphinic acid and N-methylaminomethyl-P-hydroxymethylphosphinic acid were the most effective inhibitors (Ki = 0.294 μM and 1.032 μM, respectively, compared to Ki = 23 μM for the established urease inhibitor acetohydroxamic acid). The biological relevance of the inhibitors was verified in vitro against a ureolytically active Escherichia coli Rosetta host that expressed H. pylori urease and against a reference strain, H. pylori J99 (CagA+/VacA+). The majority of the studied compounds exhibited urease-inhibiting activity in these whole-cell systems. Bis(N-methylaminomethyl)phosphinic acid was found to be the most effective inhibitor in the susceptibility profile studies of H. pylori J99. The cytotoxicity of nine structurally varied inhibitors was evaluated against four normal human cell lines and was found to be negligible.

Assessment of Sequential and Standard Triple Therapy in Treatment of Helicobacter pylori Infection in Children Dependent on Bacteria Sensitivity to Antibiotics.

BACKGROUND In the last decade a 10-day schema of sequential therapy of Helicobacter pylori infection based on proton pomp inhibitor (PPI), amoxicillin (AMO), clarithromycin (CLA) and metronidazole (MET) has been introduced. Many studies have emphasized greater efficacy of this therapy in comparison to the efficacy of the standard 7-day triple therapy (PPI + AMO + CLA or MET). OBJECTIVES The aim of the study was to assess the sequential and standard triple therapy. MATERIAL AND METHODS Sixty-nine children, aged 5 to 17 years, with symptoms of dyspepsia and gastric or duodenal ulcer were included in the study. The children were randomly divided into three groups. Group I - 23 children treated with PPI + AMO + CLA, group II - 23 children treated with PPI + AMO + MET, and group III - 23 children treated with sequential therapy. The diagnosis of Helicobacter pylori infection was based on histopathological evaluation of gastric mucosa sample and on culture. The sensitivity of bacterial strains to antibiotics was assessed based on E-tests. The efficacy of Helicobacter pylori eradication was assessed 6-8 weeks after the completion of the treatment. RESULTS In children infected with Helicobacter pylori strains, which were sensitive to clarithromycin, the highest rate of eradication was obtained in the group treated with PPI + AMO + CLA (100%) and in the group treated with sequential therapy (90.48%), the lowest was in the group treated with PPI + AMO + MET. CONCLUSIONS Efficiency of treatment of Helicobacter pylori infection in children depended on sensitivity of the strains to clarithromycin. Sensitivity to metronidazole did not influence significantly the eradication rate.

Frequency of infection with Helicobacter pylori isolates of different antimicrobial profiles in children and adolescents: A preliminary study

BACKGROUND Helicobacter pylori (H. pylori) infection can occur as a mixed infection caused by several strains of H. pylori. OBJECTIVES The aim of the study was to determine the frequency of colonization of the gastric mucosa by strains of H. pylori with different susceptibility to antimicrobial agents. MATERIAL AND METHODS The study was carried out on gastric biopsies taken from 54 previously untreated Polish children and adolescents. Of the 15 positive cultures, from each primary medium, 6 single H. pylori colonies were isolated, making a total of 90 isolates, and the susceptibility to metronidazole (MZ), amoxicillin (AC) and clarithromycin (CH) was determined by E-test method. The presence of the cagA gene and vacA alleles (s1, s2, m1, m2) was determined by PCR. RESULTS Positive culture for H. pylori was noted in 15/54 (27.7%) of patients. All H. pylori isolates were susceptible to AC, 27.8% were resistant to MZ and 38.9% to CH. The results showed 7/15 (46.7%) of children were infected with H. pylori strains with antibiotic heteroresistance, resistant to CH (5/15, 33.3%) and to MZ (2/15, 13.3%). The cagA + vacA s1/m2 combination was predominant genotype among detected H. pylori strains. The isolates possessing different antimicrobial susceptibility profiles in the same patient were identified. CONCLUSIONS Microbiological analyses confirmed the presence of isolates possessing different antimicrobial susceptibility profiles in 47% of examined children with H. pylori infection. Different antimicrobial susceptibility profiles of H. pylori isolates detected in the same patient may influence the success of eradication therapy.

Morphology of Helicobacter pylori as a result of peptidoglycan and cytoskeleton rearrangements

Helicobacter pylori is a Gram-negative, microaerophilic bacterium colonising the gastric mucosa. Normally, this bacterium has a spiral shape, which is crucial for proper colonisation of the stomach and cork-screwing penetration of dense mucin covering this organ. However, H. pylori may also form curved/straight rods, filamentous forms and coccoid forms. This morphological variability affects nutrient transport and respiration processes, as well as motility, the ability to form aggregates/biofilms, and resistance to adverse environmental factors. For this reason, a more accurate understanding of the molecular determinants that control the morphology of H. pylori seems to be crucial in increasing the effectiveness of antibacterial therapies directed against this microorganism. This article focuses on the molecular factors responsible for peptidoglycan and cytoskeleton rearrangements affecting H. pylori morphology and survivability. In addition, the existence of proteins associated with modifications of H. pylori morphology as potential targets in therapies reducing the virulence of this bacterium has been suggested.

A twelve–year retrospective analysis of prevalence and antimicrobial susceptibility patterns of Ureaplasma spp. and Mycoplasma hominis in the province of Lower Silesia in Poland

OBJECTIVE Genital mycoplasmas are opportunistic pathogens that have been associated with urogenital infections in humans. Only a few groups of antimicrobials are available for treatment of urogenital tract infections caused by genital mycoplasmas. However, emerging resistance of mycoplasmas to antimicrobial agents has been reported worldwide. The aim of the study was a retrospective analysis of the prevalence and antimicrobial susceptibility patterns of M. hominis and Ureaplasma spp. in patients with urogenital tract infections during a twelve-year period between 2003 and 2015. STUDY DESIGN Mycoplasma IST2 test was used for the detection, enumeration, identification and antimicrobial susceptibility testing of genital mycoplasmas in 1182 samples from 778 women and 404 men with genitourinary tract infection. Indicative enumeration in the test determines whether the mycoplasma count in the sample is equal or higher than the threshold set at 104 colony forming units. RESULTS A total of 152 (12.8%) samples were found to be positive for genital mycoplasmas. M. hominis was detected only in three samples and Ureaplasma spp. in 141 samples. Both, M. hominis and Ureaplasma spp. were detected in the remaining eight samples. In the analyzed period between 2003 and 2015, a gradually increasing resistance of ureaplasmas to ciprofloxacin and clarithromycin and decreasing resistance to ofloxacin, erythromycin and tetracycline were observed. Pristinamycin, josamycin and doxycycline were most active against Ureaplasma spp. In contrast, fluoroquinolones had the lowest efficacy against Ureaplasma spp. and as many as 116 (82.3%) and 77 (54.6%) of Ureaplasma spp. isolates were resistant to ciprofloxacin and ofloxacin, respectively. M. hominis isolates were uniformly resistant to azithromycin, clarithromycin and erythromycin but susceptible to josamycin, ofloxacin, doxycycline and pristinamycin. One-third of these isolates were resistant to ciprofloxacin and tetracycline. CONCLUSION In the study Ureaplasma spp. and M. hominis were detected with relatively low frequency in comparison with other studies however, most of these isolates were resistant to ciprofloxacin indicating the need for better management of ciprofloxacin prescription. Important limitations of Mycoplasma IST2 assay concerning antimicrobial susceptibility testing and divergences between breakpoints in the test and EUCAST guidelines point the need to introduce new methodologies to improve evaluation of resistant strains at our region.