Sujit Chaudhuri

Oxygen-18 stable isotope of exhaled breath CO2 as a non-invasive marker of Helicobacter pylori infection

We report for the first time the time-dependent excretion kinetics of 18O/16O isotope ratios of CO2 in exhaled breath samples using an optical cavity-enhanced integrated cavity output spectroscopy (ICOS) method for the detection of Helicobacter pylori (H. pylori) infection in human stomach. We observed large differences in the oxygen-18 isotopic fractionations of breath CO2 between H. pylori positive and negative individuals in response to orally administered, both unlabelled and labelled 13C-enriched urea, suggesting a potential link between H. pylori infections and the 18O-isotopic exchange in exhaled breath. An optimal diagnostic cut-off point of 18O/16O isotope ratios of breath CO2 for the presence of H. pylori infection was determined to be 1.92‰ using the receiver operating characteristic curve (ROC) analysis, which exhibited both diagnostic sensitivity and specificity of 100% with an accuracy of 100%. Moreover, the methodology of monitoring 18O in breath CO2 manifested both positive and negative predictive values of 100%, demonstrating excellent diagnostic accuracy and suggesting that breath C18O16O could be used as a potential marker for the identification of H. pylori infections. Our findings also suggest that monitoring the 18O/16O isotope ratios of breath CO2 is a valid and sufficiently robust novel non-invasive approach for the accurate and specific detection of H. pylori infection in real-time, which may open new perspectives in the molecular diagnosis of H. pylori infection for large-scale screening purposes, early detection and follow-up of patients.

Residual gas analyzer mass spectrometry for human breath analysis: a new tool for the non-invasive diagnosis of Helicobacter pylori infection

A residual gas analyzer (RGA) coupled with a high vacuum chamber is described for the non-invasive diagnosis of the Helicobacter pylori (H. pylori) infection through ¹³C-urea breath analysis. The present RGA-based mass spectrometry (MS) method is capable of measuring high-precision ¹³CO₂ isotope enrichments in exhaled breath samples from individuals harboring the H. pylori infection. The system exhibited 100% diagnostic sensitivity, and 93% specificity alongside positive and negative predictive values of 95% and 100%, respectively, compared with invasive endoscopy-based biopsy tests. A statistically sound diagnostic cut-off value for the presence of H. pylori was determined to be 3.0‰ using a receiver operating characteristic curve analysis. The diagnostic accuracy and validity of the results are also supported by optical off-axis integrated cavity output spectroscopy measurements. The δ¹³(DOB)C‰ values of both methods correlated well (R² = 0.9973 at 30 min). The RGA-based instrumental setup described here is simple, robust, easy-to-use and more portable and cost-effective compared to all other currently available detection methods, thus making it a new point-of-care medical diagnostic tool for the purpose of large-scale screening of the H. pylori infection in real time. The RGA-MS technique should have broad applicability for ¹³C-breath tests in a wide range of biomedical research and clinical diagnostics for many other diseases and metabolic disorders.

Diagnosis of small intestinal bacterial overgrowth in irritable bowel syndrome patients using high-precision stable 13CO2/12CO2 isotope ratios in exhaled breath

Hydrogen breath tests (HBT) are widely used for the diagnosis of small intestinal bacterial overgrowth (SIBO) in patients with irritable bowel syndrome (IBS). However, the conclusions drawn from these studies are highly controversial, and several discrepancies exist in the results. The aim of our study is to develop an alternative 13C-glucose breath test (13C-GBT) methodology by measuring high-precision 13CO2/12CO2 isotope ratios in exhaled breath to accurately diagnose SIBO using an optical cavity-enhanced CO2 isotope analyzer. In all, 118 diarrhea-predominant (IBS-D) patients diagnosed according to ROME III criteria underwent 13C-GBT and HBT following the ingestion of a test meal containing 50 mg 13C-enriched glucose with 50 g glucose. The excretion of 13CO2 enrichments and a cumulative percentage dose of 13C-recovered (c-PDR) significantly depleted (p < 0.001) for the positive SIBO patients (n = 25) compared to the patients with negative SIBO (n = 53) as diagnosed by HBT. A cut-off value of 5.47‰ at 45 min was indicative of a positive SIBO syndrome. Subsequently, a portion of IBS-D individuals (n = 20) whose HBTs were negative but 13C-GBTs were positive, suggesting HBT often fails to diagnose SIBO when the patients may have “non-hydrogen-producing” bacteria. 13C-GBT also correctly diagnosed SIBO in patients (n = 20) within the “grey-zone” and during the preclinical phase of SIBO as opposed to HBT. The prevalence of SIBO with IBS-D in Indian population was estimated to be 45.7%. Our study demonstrates 13C-GBT, for the first time, as a clinically valid and sufficiently robust alternative diagnostic methodology for an accurate evaluation of SIBO in IBS-D patients and superior to HBT.

Mechanisms linking metabolism of Helicobacter pylori to 18O and 13C-isotopes of human breath CO2

The gastric pathogen Helicobacter pylori utilize glucose during metabolism, but the underlying mechanisms linking to oxygen-18 (18O) and carbon-13 (13C)-isotopic fractionations of breath CO2 during glucose metabolism are poorly understood. Using the excretion dynamics of 18O/16O and 13C/12C-isotope ratios of breath CO2, we found that individuals with Helicobacter pylori infections exhibited significantly higher isotopic enrichments of 18O in breath CO2 during the 2h-glucose metabolism regardless of the isotopic nature of the substrate, while no significant enrichments of 18O in breath CO2 were manifested in individuals without the infections. In contrast, the 13C-isotopic enrichments of breath CO2 were significantly higher in individuals with Helicobacter pylori compared to individuals without infections in response to 13C-enriched glucose uptake, whereas a distinguishable change of breath 13C/12C-isotope ratios was also evident when Helicobacter pylori utilize natural glucose. Moreover, monitoring the 18O and 13C-isotopic exchange in breath CO2 successfully diagnosed the eradications of Helicobacter pylori infections following a standard therapy. Our findings suggest that breath 12C18O16O and 13C16O16O can be used as potential molecular biomarkers to distinctively track the pathogenesis of Helicobacter pylori and also for eradication purposes and thus may open new perspectives into the pathogen’s physiology along with isotope-specific non-invasive diagnosis of the infection.

Natural 18 O and 13 C-urea in gastric juice: a new route for non-invasive detection of ulcers

AbstractThe 13C-urea breath test (13C-UBT), developed a few decades ago, is widely used as a non-invasive diagnostic method to detect only the presence of the gastric pathogen Helicobacter pylori infection; however, the actual disease state, i.e. whether the person harbouring H. pylori has peptic ulcer disease (PUD) or non-ulcerous dyspepsia (NUD), is still poorly understood. Nevertheless, the present 13C-UBT has numerous limitations, drawbacks and pitfalls owing to the ingestion of 13C-labelled external urea. Here, we show that H. pylori is able to utilize the natural 13C and 18O-urea inherently present in the gastric juice in humans for its urease activity which has never been explored before. In vitro measurements of isotopic fractionations of gastric juice urea provide new insights into the actual state of the infection of PUD or NUD. We also provide evidence of the unusual 13C and 18O-isotopic fractionations of breath CO2 that are distinctively altered in individuals with PUD encompassing both gastric and duodenal ulcers as well as with NUD by the enzymatic activity of H. pylori in the gastric niche without oral administration of any 13C-enriched external urea. This deepens our understanding of the UBT exploiting the natural 13C and 18O-gastric juice urea in the pathogenesis of H. pylori infection, reveals the actual disease state of PUD or NUD and thus offers novel opportunities for a simple, robust, cost-effective and non-toxic global strategy devoid of any 13C-enriched urea for treating these common diseases by a single breath test. Graphical AbstractUrea breath test without any external urea

Molecular hydrogen in human breath: a new strategy for selectively diagnosing peptic ulcer disease, non-ulcerous dyspepsia and Helicobacter pylori infection

The gastric pathogen Helicobacter pylori utilizes molecular hydrogen (H2) as a respiratory substrate during colonization in the gastric mucosa. However, the link between molecular H2 and the pathogenesis of peptic-ulcer disease (PUD) and non-ulcerous dyspepsia (NUD) by the enzymatic activity of H. pylori still remains mostly unknown. Here we provide evidence that breath H2 excretion profiles are distinctly altered by the enzymatic activity of H. pylori for individuals with NUD and PUD. We subsequently unravelled the potential molecular mechanisms responsible for the alteration of H2 in exhaled breath in association with peptic ulcers, encompassing both gastric and duodenal ulcers, along with NUD. We also established that carbon-isotopic fractionations in the acid-mediated bacterial environment regulated by bacterial urease activity cannot discriminate the actual disease state i.e. whether it is peptic ulcer or NUD. However, our findings illuminate the unusual molecular H2 in breath that can track the precise evolution of PUD and NUD, even after the eradication of H. pylori infection. This deepens our understanding of the pathophysiology of PUD and NUD, reveals non-invasively the actual disease state in real-time and thus offers a novel and robust new-generation strategy for treating peptic-ulcer disease together with non-ulcer related complications even when the existing (13)C-urea breath test ((13)C-UBT) fails to diagnose.

Exploring the physiological link of breath N 2 O through nitrification and denitrification processes in human gastric juice

Over the past several decades, it has been generally believed that microbial nitrification and denitrification are not significant processes in the human gastrointestinal tract. Moreover, the underlying physiological link between exhaled nitrous oxide (N2O) and aerobic denitrification in the gastric environment is still largely unknown. In this report, we provide direct experimental evidence of the aerobic denitrification process in the human gastrointestinal tract by evaluating concentrations of dissolved N2O and its precursor nitrite ([Formula: see text]) ion in the gastric juice along with exhaled N2O concentration using a high-precision laser spectroscopy technique. Moreover, in vitro studies of gastric fluid in patients reveal a new mechanism of nitrification of ammonium ion ([Formula: see text]) followed by denitrification of [Formula: see text] leading to the formation of N2O in the gastric environment, which is eventually excreted in exhaled breath. This observation was subsequently validated under in vivo physiological conditions exploiting the urease activity of the gastric pathogen Helicobacter pylori. Consequently, our findings established a strong physiological link between exhaled N2O and bacterial infection in the stomach. This deepens our understanding of the unusual microbial denitrification in the gastric environment, providing new insight into the activities of human-associated microorganisms, which eventually affect the human physiology and health.

Exhaled nitric oxide as a potential marker for detecting non-ulcer dyspepsia and peptic ulcer disease

Nitric oxide (NO) plays a key role in the development of peptic ulcer disease (PUD). Conversely, the gastric pathogen Helicobacter pylori colonizes the human stomach and contributes to the development of non-ulcer dyspepsia (NUD) and PUD. However, the underlying relation between molecular NO in exhaled breath and H. pylori-associated NUD and PUD remains largely unknown. Here, we found that the excretion kinetics of NO profiles in exhaled breath are altered markedly in H. pylori-infected NUD and PUD subjects. In our observations, PUD led to considerably higher enrichments of NO in exhaled breath compared to NUD, thus revealing a potential link between exhaled NO and ulcer and non-ulcer complications. Our findings therefore suggest that molecular NO in exhaled breath could be used as a potential biomarker for non-invasive diagnosis and selective differentiation of NUD from PUD. Our observations also highlight that alterations of NO in the gastric environment can play an important role in the pathogenesis of peptic ulcers and thus may provide a new strategy for precise evolution of the actual disease state without the need for endoscopic biopsy, even after the eradication of H. pylori infection.

Non-invasive diagnosis of type 2 diabetes in Helicobacter pylori infected patients using isotope-specific infrared absorption measurements

ABSTRACT Helicobacter pylori causes several gastrointestinal diseases and may also contribute to the development of type 2 diabetes (T2D). Several studies suggest that there might be a potential link between H. pylori infection and T2D, but it still remains the subject of debate. Here, we first report the cumulative effect of H. pylori infection and T2D by exploiting the excretion kinetics of 13C/12C and 18O/16O isotope ratios of exhaled breath CO2 in response to an oral dose of 13C-enriched glucose in individuals with T2D and non-diabetic controls (NDC) harbouring the H. pylori infection. Using a high-resolution integrated cavity output spectroscopy (ICOS) technique in the infrared region, we observed that the isotopic fractionations of 13C and 18O in breath CO2 are distinctly altered in H. pylori infected T2D patients as well as in H. pylori infected NDC. Several optimal diagnostic cut-off points of 13C and 18O isotopes of breath CO2 were also determined which exhibited the diagnostic sensitivity and specificity of ∼97 % and thus suggesting that breath 13C and 18O isotopes might be considered as potential biomarkers for the non-invasive assessment of the gastric pathogen prior to the onset of T2D. This may open a new diagnostic strategy for treating these common diseases in an alternative way.