Volatilomic signatures of different strains of Helicobacter pylori.

BACKGROUND: Helicobacter pylori (H. pylori) infection is the most extensively studied risk factor for gastric cancer. As with any bacteria, H. pylori will release distinctive odors that result from an emission of volatile metabolic byproducts in unique combinations and proportions. Effectively capturing and identifying these volatiles can pave the way for the development of innovative and non-invasive diagnostic methods for determining infection. Here we characterize the H. pylori volatilomic signature, pinpoint potential biomarkers of its presence, and evaluate the variability of volatilomic signatures between different H. pylori isolates. MATERIALS AND METHODS: Using needle trap extraction, volatiles in the headspace above H. pylori cultures were collected and, following thermal desorption at 290°C in a splitless mode, were analyzed using gas chromatography-mass spectrometry. The resulting volatilomic signatures of H. pylori cultures were compared to those obtained from an analysis of the volatiles in the headspace above the cultivating medium only. RESULTS: Amongst the volatiles detected, 21 showed consistent differences between the bacteria cultures and the cultivation medium, with 11 compounds being elevated and 10 showing decreased levels in the culture's headspace. The 11 elevated volatiles are four ketones (2-pentanone, 5-methyl-3-heptanone, 2-heptanone, and 2-nonanone), three alcohols (2-methyl-1-propanol, 3-methyl-1-butanol, and 1 butanol), one aromatic (styrene), one aldehyde (2-ethyl-hexanal), one hydrocarbon (n-octane), and one sulfur compound (dimethyl disulfide). The 10 volatiles with lower levels in the headspace of the cultures are four aldehydes (2-methylpropanal, benzaldehyde, 3-methylbutanal, and butanal), two heterocyclic compounds (2-ethylfuran and 2-pentylfuran), one ketone (2-butanone), one aromatic (benzene), one alcohol (2-butanol) and bromodichloromethane. Of the volatile species showing increased levels, the highest emissions are found to be for 3-methyl-1-butanol, 1-butanol and dimethyl disulfide. Qualitative variations in their emissions from the different isolates was observed. CONCLUSIONS: The volatiles emitted by H. pylori provide a characteristic volatilome signature that has the potential of being developed as a tool for monitoring infections caused by this pathogen. Furthermore, using the volatilome signature, we are able to differentiate different isolates of H. pylori. However, the volatiles also represent potential confounders for the recognition of gastric cancer volatile markers.

Randomised clinical trial: efficacy and safety of H. pylori eradication treatment with and without Saccharomyces boulardii supplementation.

BACKGROUND: Standard triple therapy is commonly prescribed Helicobacter pylori eradication regimen in Europe. However, the world is witnessing declines in eradication success. It is crucial to find better treatment options. AIMS: To evaluate efficacy, compliance and side effects of H. pylori eradication treatment by adding Saccharomyces boulardii . METHODS: We conducted a randomized clinical trial within the GISTAR cohort, consisting of healthy individuals aged 40-64 years. Participants were administered clarithromycin-containing triple therapy (clarithromycin 500 mg, amoxicillin 1000 mg, esomeprazole 40 mg) twice daily. Randomization was applied based on two factors: 1)addition of Saccharomyces boulardii CNCM I-745 500 mg BID or not; 2)treatment duration of 10 or 14 days. Treatment completion and adverse events were assessed via telephone interview 21-28 days after medication delivery. The efficacy was evaluated using a 13C-urea breath test (UBT) six months after treatment. RESULTS: Altogether 404 participants were enrolled; data on adverse events were available from 391. Overall, 286 participants received follow-up UBT. Intention-to-treat analysis revealed higher eradication rates for 10-day probiotic treatment (70.8% vs. 54.6%, P  = 0.022), but not for 14-day. Probiotic subgroups combined showed non-significantly higher efficacy in per-protocol analysis (90.6% vs. 85.0%, P  = 0.183). S. boulardii reduced the frequency of adverse events ( P  = 0.033) in 14-day regimen, particularly treatment-associated diarrhea ( P  = 0.032). However, after the adjustment to control Type I error, results lost their significance. CONCLUSION: Addition of S. boulardii to 14-day clarithromycin-containing triple regimen non-significantly lowers the likelihood of diarrhea and does not increase the eradication rate.

Multiplexed RT-qPCR Coupled with Whole-Genome Sequencing to Monitor a SARS-CoV-2 Omicron Variant of Concern in a Hospital Laboratory Setting in Latvia.

At the end of 2021, the SARS-CoV-2 Omicron variant of concern (VOC) displaced the previously dominant Delta VOC and enhanced diagnostic and therapeutic challenges worldwide. Respiratory specimens submitted to the Riga East University Hospital Laboratory Service by the central and regional hospitals of Latvia from January to March 2022 that were positive for SARS-CoV-2 RNA were tested by commercial multiplexed RT-qPCR targeting three of the Omicron VOC signature mutations: ΔH69/V70, E484A, and N501Y. Of the specimens tested and analyzed in parallel by whole-genome sequencing (WGS), 964 passed the internal quality criteria (genome coverage ≥90%, read depth ≥400×) and the Nextstrain's quality threshold for "good". We validated the detection accuracy of RT-qPCR for each target individually by using WGS as a control. The results were concordant with both approaches for 938 specimens, with the correct classification rate exceeding 96% for each target (CI 95%); however, the presumptive WHO label was misassigned for 21 specimens. The RT-qPCR genotyping provided an acceptable means to pre-monitor the prevalence of the two presumptive Omicron VOC sublineages, BA.1 and BA.2.

Abundance and prevalence of ESBL coding genes in patients undergoing first line eradication therapy for Helicobacter pylori.

The spread of extended-spectrum beta-lactamases (ESBLs) in nosocomial and community-acquired enterobacteria is an important challenge for clinicians due to the limited therapeutic options for infections that are caused by these organisms. Here, we developed a panel of ESBL coding genes, evaluated the abundance and prevalence of ESBL encoding genes in patients undergoing H. pylori eradication therapy, and summarized the effects of eradication therapy on functional profiles of the gut microbiome. To assess the repertoire of known beta lactamase (BL) genes, they were divided into clusters according to their evolutionary relation. Primers were designed for amplification of cluster marker regions, and the efficiency of this amplification panel was assessed in 120 fecal samples acquired from 60 patients undergoing H. pylori eradication therapy. In addition, fecal samples from an additional 30 patients were used to validate the detection efficiency of the developed ESBL panel. The presence for majority of targeted clusters was confirmed by NGS of amplification products. Metagenomic sequencing revealed that the abundance of ESBL genes within the pool of microorganisms was very low. The global relative abundances of the ESBL-coding gene clusters did not differ significantly among treatment states. However, at the level of each cluster, classical ESBL producers such as Klebsiella sp. for blaOXY (p = 0.0076), Acinetobacter sp. for blaADC (p = 0.02297) and others, differed significantly with a tendency to decrease compared to the pre- and post-eradication states. Only 13 clusters were common across all three datasets, suggesting a patient-specific distribution profile of ESBL-coding genes. The number of AMR genes detected in the post-eradication state was higher than that in the pre-eradication state, which could be attributed, at least in part, to the therapy. This study demonstrated that the ESBL screening panel was effective in targeting ESBL-coding gene clusters from bacterial DNA and that minor differences exist in the abundance and prevalence of ESBL-coding gene levels before and after eradication therapy.

Volatile Markers for Cancer in Exhaled Breath-Could They Be the Signature of the Gut Microbiota?

It has been shown that the gut microbiota plays a central role in human health and disease. A wide range of volatile metabolites present in exhaled breath have been linked with gut microbiota and proposed as a non-invasive marker for monitoring pathological conditions. The aim of this study was to examine the possible correlation between volatile organic compounds (VOCs) in exhaled breath and the fecal microbiome by multivariate statistical analysis in gastric cancer patients (n = 16) and healthy controls (n = 33). Shotgun metagenomic sequencing was used to characterize the fecal microbiota. Breath-VOC profiles in the same participants were identified by an untargeted gas chromatography-mass spectrometry (GC-MS) technique. A multivariate statistical approach involving a canonical correlation analysis (CCA) and sparse principal component analysis identified the significant relationship between the breath VOCs and fecal microbiota. This relation was found to differ between gastric cancer patients and healthy controls. In 16 cancer cases, 14 distinct metabolites identified from the breath belonging to hydrocarbons, alcohols, aromatics, ketones, ethers, and organosulfur compounds were highly correlated with 33 fecal bacterial taxa (correlation of 0.891, p-value 0.045), whereas in 33 healthy controls, 7 volatile metabolites belonging to alcohols, aldehydes, esters, phenols, and benzamide derivatives correlated with 17 bacterial taxa (correlation of 0.871, p-value 0.0007). This study suggested that the correlation between fecal microbiota and breath VOCs was effective in identifying exhaled volatile metabolites and the functional effects of microbiome, thus helping to understand cancer-related changes and improving the survival and life expectancy in gastric cancer patients.

Liquid Biopsy-Based Volatile Organic Compounds from Blood and Urine and Their Combined Data Sets for Highly Accurate Detection of Cancer.

Liquid biopsy is seen as a prospective tool for cancer screening and tracking. However, the difficulty lies in effectively sieving, isolating, and overseeing cancer biomarkers from the backdrop of multiple disrupting cells and substances. The current study reports on the ability to perform liquid biopsy without the need to physically filter and/or isolate the cancer cells per se. This has been achieved through the detection and classification of volatile organic compounds (VOCs) emitted from the cancer cells found in the headspace of blood or urine samples or a combined data set of both. Spectrometric analysis shows that blood and urine contain complementary or overlapping VOC information on kidney cancer, gastric cancer, lung cancer, and fibrogastroscopy subjects. Based on this information, a nanomaterial-based chemical sensor array in conjugation with machine learning as well as data fusion of the signals achieved was carried out on various body fluids to assess the VOC profiles of cancer. The detection of VOC patterns by either Gas Chromatography-Mass Spectrometry (GC-MS) analysis or our sensor array achieved >90% accuracy, >80% sensitivity, and >80% specificity in different binary classification tasks. The hybrid approach, namely, analyzing the VOC datasets of blood and urine together, contributes an additional discrimination ability to the improvement (>3%) of the model's accuracy. The contribution of the hybrid approach for an additional discrimination ability to the improvement of the model's accuracy is examined and reported.

Assessing the utility of pepsinogens and gastrin-17 in gastric cancer detection.

OBJECTIVES: The aim of the study was to determine the proportion of gastric cancer patients with decreased levels of pepsinogen and gastrin-17 in plasma, with the goal of providing indirect evidence of the sensitivity of these biomarkers when applied in a cancer screening setting. METHODS: The levels of pepsinogens I and II, gastrin-17, and Helicobacter pylori immunoglobulin antibodies in plasma samples of gastric cancer patients were evaluated using the GastroPanel test system (Biohit Oyj, Helsinki, Finland). A decreased level of the pepsinogen I/II ratio was defined as less than three, while a decrease in gastrin-17 was defined as less than 1 pmol/L. Univariate analysis using non-parametric tests was used to investigate differences between normal and low concentrations of biomarkers. RESULTS: In total, 481 plasma samples from patients (59.9% male) with a median age of 64 years (ranging from 27 to 88 years) were analyzed. Out of the 400 cases of gastric cancer (83.2% of the total), 182 were categorized as the intestinal type, 141 as the diffuse type, 60 as the mixed type, and 17 as indeterminate according to the Lauren classification system. The H. pylori immunoglobulin test was positive in 74.0% of the patients. Pepsinogen I/II ratio was decreased in 32.4% (36.8% of the intestinal type); gastrin-17 in 12.3% (10.1% of the antral region) of all cases. CONCLUSION: The majority of gastric cancer patients had normal levels of pepsinogen and gastrin-17, suggesting that these biomarkers have limited application as screening tools in the Caucasian population.

Factors Associated with False Negative Results in Serum Pepsinogen Testing for Precancerous Gastric Lesions in a European Population in the GISTAR Study.

The accuracy of plasma pepsinogen (Pg) as a marker for precancerous gastric lesions (PGL) has shown variable results. We aimed to identify factors associated with false negative (FN) cases in Pg testing and to adjust cut-off values for these factors in order to improve Pg yield. Plasma Pg was measured and upper endoscopy with biopsy was performed within the "Multicentric randomized study of Helicobacter pylori eradication and pepsinogen testing for prevention of gastric cancer mortality: the GISTAR study". A multivariable logistic model was built for FN and multiple factors. Values of Pg were compared and sensitivity and specificity were calculated using pre-existing Pg cut-offs for factors showing strong associations with FN. New cut-offs were calculated for factors that showed substantially lower sensitivity. Of 1210 participants, 364 (30.1%) had histologically confirmed PGL, of which 160 (44.0%) were FN. Current smokers, men, and H. pylori positives were more likely FN. Smoking in H. pylori negatives was associated with a higher Pg I/II ratio and substantially lower sensitivity of Pg testing than in other groups. Adjusting Pg cut-offs for current smokers by H. pylori presence improved sensitivity for detecting PGL in this group. Our study suggests that adjusting Pg cut-offs for current smokers by H. pylori status could improve Pg test performance.

Randomised clinical trial: comparison of efficacy and adverse effects of a standard triple clarithromycin-containing regimen with high-dose amoxicillin and bismuth therapy in Helicobacter pylori eradication.

BACKGROUND: The clarithromycin-based triple therapy is the most prescribed Helicobacter pylori eradication regimen in Europe; it causes adverse effects in a significant proportion of subjects, leading to discontinuation. Alternative therapies are required because of increasing clarithromycin resistance or to decrease the adverse effects. AIMS: We compared the efficacy and spectrum of adverse effects of clarithromycin-based triple therapy with the high-dose amoxicillin/bismuth regimen. METHODS: A randomised clinical trial enrolled healthy individuals aged 40-64 years. H. pylori was assessed with a 13C-urea breath test. In total 579 H. pylori-positive subjects were randomly allocated in two groups: group 1: clarithromycin 500 mg, amoxicillin 1000 mg, esomeprazole 40 mg, all twice daily; group 2: bismuth subcitrate 240 mg twice daily, amoxicillin 1000 mg three times daily, esomeprazole 40 mg twice daily. Regimens were administered for 14 days.Information on treatment completion and adverse effects were collected via a telephone interview at 21-28 days after medication delivery. The efficacy was assessed by UBT 6 months after the treatment. RESULTS: We analysed 483 subjects for adverse effects (248 vs. 235 respectively). Furthermore, 316 subjects were analysed for efficacy. In per-protocol analysis, a higher efficacy was seen in group 1 (88.4 vs. 77.0%; P < 0.001); no difference was observed in compliance (90.3 and 91.2%). Therapy-related adverse effects were more common in group 1 (56.9 vs. 40.0%; P < 0.01). In intention-to-treat analysis no statistical difference in efficacy was revealed. CONCLUSIONS: Bismuth-based high-dose amoxicillin therapy showed a lower efficacy but was less frequently associated with adverse effects. Further research is required to examine the high-dose amoxicillin and bismuth-containing regimens in various populations to maximise eradication efficacy.

Lifestyle and dietary factors associated with serologically detected gastric atrophy in a Caucasian population in the GISTAR study.

OBJECTIVE: To identify dietary and lifestyle factors associated with decreased pepsinogen levels indicative of gastric atrophy. METHODS: Participants aged 40 to 64 from the "Multicentric randomized study of H. pylori eradication and pepsinogen testing for prevention of gastric cancer mortality (GISTAR study)" in Latvia tested for serum pepsinogen, as well as for Helicobacter pylori infection by 13 C-urea breath test or serology were included. Data on sex, age, education, employment, diet, smoking, alcohol and proton pump inhibitor use were obtained by survey and compared for participants with and without serologically detected gastric atrophy defined as pepsinogen I/pepsinogen II ≤ 2 and pepsinogen I ≤ 30 ng/mL. RESULTS: Of 3001 participants (median age 53, interquartile range, 11.0, 36.9% male) 52.8% had H. pylori and 7.7% had serologically detected gastric atrophy. In multivariate analysis, increasing age, consumption of alcohol, coffee, and onions were positively, while H. pylori , former smoking, pickled product and proton pump inhibitor use were inversely associated with gastric atrophy. Pepsinogen values were higher in smokers and those with H. pylori . Pepsinogen ratio was lower in those with H. pylori . When stratifying by H. pylori presence, significantly higher pepsinogen levels remained for smokers without H. pylori . CONCLUSION: Several dietary factors and smoking were associated with serologically detected gastric atrophy. Pepsinogen levels differed by smoking and H. pylori status, which may affect the serologic detection of gastric atrophy. There seems to be a complicated interaction between multiple factors. A prospective study including atrophy determined by both serology and histology is necessary.

Who Could Be Blamed in the Case of Discrepant Histology and Serology Results for Helicobacter pylori Detection?

BACKGROUND: Discrepancies between histology and serology results for Helicobacter pylori detection could be caused by a variety of factors, including a biopsy sampling error, expertise of the pathologist, natural loss of infection due to advanced atrophy, or a false-positive serology in the case of a previous infection, since antibodies may be present in blood following recovery from the infection. AIMS: To identify true H. pylori-positive individuals in discrepant cases by serology and histology using real time polymerase chain reaction (RT-PCR) as a gold standard. METHODS: Study subjects with discrepant histology and serology results were selected from the GISTAR pilot study data base in Latvia. Subjects having received previous H. pylori eradication therapy or reporting use of proton pump inhibitors, antibacterial medications, or bismuth containing drugs one month prior to upper endoscopy were excluded. We compared the discrepant cases to the corresponding results of RT-PCR performed on gastric biopsies. RESULTS: In total, 97 individuals with discrepant results were identified: 81 subjects were serology-positive/histology-negative, while 16 were serology-negative/histology-positive. Among the serology-positive/histology-negative cases, 64/81 (79.0%) were false-positives by serology and, for the majority, inflammation was absent in all biopsies, while, in the serology-negative/histology-positive group, only 6.2% were proven false-positives by histology. CONCLUSIONS: Among this high H. pylori prevalent, middle-aged population, the majority of discrepant cases between serology and histology were due to false positive-serology, rather than false-negative histology. This confirms the available evidence that the choice of treatment should not be based solely on the serological results, but also after excluding previous, self-reported eradication therapy.

Prevalence and Potential Risk Factors of Helicobacter pylori Infection among Asymptomatic Individuals in Kazakhstan.

BACKGROUND: Helicobacter pylori (H. pylori) infection is associated with several risk factors such as demographic, socioeconomic status and personal habits, which vary in different populations. This is the most up-to-date data on H. pylori prevalence and potential risk factors for H. pylori infection among asymptomatic middle-aged individuals in Kazakhstan. METHODS: Apparently healthy individuals aged 40 to 64, who took part in the health control in the outpatient clinic, were invited to participate in the study; answered a questionnaire, donated a blood sample. The antibodies to H. pylori were analysed by latex agglutination method. The baseline characteristics of study subjects with or without H. pylori infection were compared using the Chi-square test. Odds ratio (OR) and 95% confidence intervals (CI) for the association between H. pylori infection and potential risk factors were estimated using multivariable logistic regression models. RESULTS: Altogether 166 subjects (59% male; the median age - 51 years old) were included; 104 (62.7%) were H. pylori positive. There were no statistically significant differences between H. pylori positive and H. pylori negative groups in respect to the gender, anthropometric measurements, socioeconomic factors and personal habits. The multiple variable analysis showed that age (OR, 1.99; 95% CI, 1.03 - 3.86; P=0.04) and increased salt intake (OR, 2.21; 95% CI, 1.12 - 4.35; P=0.02) were associated with H. pylori infection. CONCLUSIONS: More than half of the study subjects were infected with H. pylori in Kazakhstan. The prevalence of H. pylori infection was independently associated with older age and regular high salt consumption.

Lack of significant differences between gastrointestinal tract microbial population structure of Helicobacter pylori-infected subjects before and 2 years after a single eradication event.

BACKGROUND: According to recent estimates 80% of Latvian population is infected with Helicobacter pylori thus their susceptibility to numerous gastric tract diseases is increased. The 1st line H. pylori eradication therapy includes treatment with clarithromycin in combination with amoxicillin or metronidazole and a proton pump inhibitor. However, potential adverse events caused by such therapies to microbiome are insufficiently studied. OBJECTIVE: This study aimed to evaluate the long-term effect of H. pylori eradication on human gastrointestinal tract (GIT) microbiome. METHODS: The assessment of H pylori eradication impact on GIT microbiome was done by analyzing 120 samples acquired from 60 subjects. Each individual was prescribed the following 10-day eradication regimen: Esomeprazolum 40 mg, Clarithromycinum 500 mg, and Amoxicillinum 1000 mg, BID. Samples from each individual were collected before starting H pylori eradication therapy, and 2 years after the completion of the therapy in OC-Sensor (Eiken Chemical Co.) sample collection containers and stored at -86°C. Prior to DNA extraction, the samples were lyophilized, and total DNA was extracted using FastDNA Spin Kit for Soil. 16S V3 rRNA gene sequencing was done employing Ion Torrent PGM, and the obtained raw sequences were analyzed using vsearch and R (phyloseq, cluster packages). RESULTS: Alpha diversity measurements-observed OTUs, Chao1 and Shannon index did not differ significantly between the pre- and post-eradication states (two-tailed paired t test: P = .95; P = .71, P = .24, respectively). Unweighted and weighted UniFrac distances of beta diversity analysis indicated a non-specific pattern of sample clustering. Enterotype shift was observed for the majority of individuals comparing pre- and post-eradication study groups. Association analysis revealed that certain bacterial genera significantly correlated with age (eg, Dialister, Paraprevotella, Bifidobacterium), individual (eg, Thermotunica, Streptomyces, Faecalibacterium), and history of respiratory and/or allergic diseases (eg, Colinsella, Faecalibacterium). Redundancy analysis confirmed that the individual was a significant determinant of the subject's microbial community composition (ANOVA, 999 perm., P = .001) with the further lower impact of subject-specific medical history (eg, medication used as prescribed: P = .005, history of cardiovascular diseases: P = .005, history of respiratory, and/or allergic diseases: P = .015) and physiological (eg, age: P = .005, gender: P = .02) parameters. In the post-eradication study group, number of influential genera (n = 260) was increased compared to the pre-eradication study group (n = 209). CONCLUSION: Modest global differences at the community level exist between individuals before and after the eradication therapy; however, the microbiome structure is more related to the subject-specific parameters rather than by the eradication therapy itself.