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Helicobacter pylori infection has been incriminated for many diseases of the upper gastrointestinal tract[1] and is also believed to be associated with many other non-gastric diseases.[2] It is estimated that more than half of the adult population in the developed world and more than 80% of that in the developing countries is infected with this bacterium.[3,4]

Conventionally the ¹⁴C-urea breath test (UBT) with its high diagnostic accuracy (>95%) is employed worldwide for the detection of H. pylori infection, for follow-up after eradication of infection and for epidemiological purposes. A large body of data leading to this conclusion has been recently considered by the Maastricht Consensus Report which concurred that UBT is a simple, non-invasive, practical, highly accurate and reproducible test of choice for the confirmation of H. pylori infection.[5] The main advantage of this test is that it samples the whole stomach and thus prevents any sampling error because of patchy distribution of these microorganisms in the stomach.[6]

Marshall and Surveyor in 1988[7] introduced a non-capsulated liquid based ¹⁴C -UBT for detection of active H. pylori infection. To avoid the problem of ¹⁴C-urea hydrolysis in the oropharynx owing to the presence of urease producing microorganisms, this test was subsequently modified by Hamlet and his coworkers in 1995.[8] These authors recommended the administration of the tracer (¹⁴C-urea) in a gelatin capsule to prevent the release of ¹⁴C-urea before reaching the stomach and to avoid overlapping results between infected and noninfected patients. At present majority of diagnostic centers employ the capsulated ¹⁴C-UBT protocol[9-11] while others use the non-capsulated protocol.[12,13]

Till date, only two reports have been published in literature which compared the data of capsulated versus non-capsulated ¹⁴C-UBT protocols in H. pylori infected and non-infected subjects.[8,14] The major limitation of these studies were that the authors did not instruct the subjects to brush their teeth and rinse their mouths before performing the non-capsulated ¹⁴CUBT test, a prerequisite strongly advocated by the earlier workers.[7,15] In addition, the test conditions were not identical to facilitate comparison of these two ¹⁴C-UBT protocols.[14] No study so far has been done to monitor the movement of [14]Curea containing capsule after its ingestion and to know the extent of its dissolution in the stomach while collecting the breath samples.

Considering the above lacunae, we developed a novel design for our study by employing a ¹⁴C-UBT-cum-scintigraphy technique to simultaneous monitor the real-time movement of the capsule in the upper gastric tract while performing the ¹⁴CUBT. The aim of our preliminary study was to compare the results of capsulated versus non-capsulated ¹⁴C-UBT protocols in H. pylori infected patients. Our studies demonstrate how sometimes H. pylori infection can be misdiagnosed due to either rapid transit of the 14C-urea containing capsule from the upper gastric tract or its incomplete resolution in the stomach during the phase of breath collection after ingestion. In this article we present three cases which were misdiagnosed for H. pylori infection using the capsulated protocol but were confirmed on using the non-capsulated method.

Methods

Subjects

Fifty eight patients (41 males; 17 females; mean age 40.05 ± 10.86 years; range 19-71 years) with upper gastrointestinal symptoms referred from Gastroenterology department of Post Graduate Institute of Medical Education and Research, Chandigarh, India were included in this study. Upper GI endoscopy was done and multiple biopsies were obtained from each patient for rapid urease test (RUT) and histopathology examination. None of these patients had taken any drugs that interfered with the UBT results.[16] The Ethics Committee of the Institute cleared the study and each of these patients gave written informed consent. ¹⁴C-urea breath test (¹⁴C-UBT): Non-capsulated UBT ¹⁴C-urea of high specific activity (1.85 GBq/mmol, Amersham, UK) was dissolved in triple distilled autoclaved water, dispensed in small sterilized vials and stored at -20°C till further use.

Non-capsulated ¹⁴C-UBT was performed as reported earlier[17] and the patient was prepared for the procedure as described elsewhere.[15] Briefly, overnight fasting patients were asked to thoroughly brush their teeth and rinse their mouths to get rid of oral urease producing commensal flora just before starting the UBT. Each patient was given 74 kBq of ¹⁴C-urea (0.4 mL) in 10 mL of water with the instruction to swallow it immediately followed by additional 250 mL of drinking water. One breath sample was obtained before ingestion of ¹⁴C-urea (to serve as background) and others at 10, 15 and 20 min directly into a CO2 trapping solution containing 0.5 mmol ethanolic benzethonium hydroxide in 10 mL toluene-based scintillation fluid along with a drop of alcoholic phenolphthalein as a pH indicator. Breath samples were collected until the color of the trapping solution changed from pink to colorless. Radioactivity in breath samples was measured as disintegration per minute (DPM) mode in liquid scintillation counter (Wallac 1409, Finland). The results were expressed as ¹⁴CO2/mmol CO2 exhaled as percent of administered ¹⁴C-urea dose and a value higher than 0.006% at any one time point was considered as an indicator of H. pylori infection.[18]

Capsulated UBT

14C-UBT study was repeated within 2 days of the first breath test using 0.5 mL capacity pharmaceutical grade gelatin capsule (M/S Parke Davis, USA). A background breath sample was obtained in trapping solution before starting the ¹⁴C-UBT. Each patient was given 74 kBq (0.4 mL) of ¹⁴C-urea along with 6.0 MBq (0.1 mL) of 99mTc-diethylene triamine penta-acetic acid (99mTc-DTPA) in a capsule. The capsule was prepared in less than 15 sec to maintain its strength. The patient was instructed to swallow the capsule immediately with 250 mL of drinking water. Breath samples were obtained up to 20 min as stated earlier and radioactivity was measured. 99mTc-DTPA being a non-absorbable marker was used in the capsule to monitor its real time movement in the stomach.[19]

Scintiscanning

Prior to conducting the capsulated ¹⁴C-UBT, the stomach contour of each patient was delineated as baseline reference for monitoring the fate of capsule. For this, each overnight fasting patient was initially injected intravenously 20.0 MBq of 99mTechnetium pertechnetate (99mTcO4 -). After half an hour static anterior scintiscan images of upper abdomen were obtained for one min on gamma camera (dual head singlephoton emission computed tomography; E-Cam, Siemens, Germany) with the patient lying in supine position. Thereafter the patient was administered a gelatin capsule containing ¹⁴Curea and 99mDTPA orally, as stated earlier. Immediately dynamic scintiscanning of anterior aspect of abdomen was done for 23 min and images were acquired at the rate of one frame per min. During acquisition of the images, breath samples were also collected.

Results

Out of 58 H. pylori infected cases (confirmed with histological evaluation, RUT and non-capsulated ¹⁴C-UBT protocol), 3 (5.17%) were found to be negative for H. pylori using capsulated the ¹⁴C-UBT protocol. Table 1 shows discriminate ¹⁴C-UBT values with and without encapsulation of ¹⁴C-urea in these cases. By employing the non-capsulated protocol, we observed that the breath ¹⁴CO2 values in these patients were higher than the cut-off value at two or more time points; whereas by using the capsulated method the test values remained low at all the time points.

In this study, we showed real time movement of the ¹⁴Curea containing capsule in the upper gastric tract with scintigraphic imaging using 99mDTPA as a gastric emptying marker. These images revealed that the misdiagnosis of H. pylori infection occurred mainly due to either rapid transit of the capsule from the upper gastric tract or its incomplete resolution in the stomach during the phase of breath collection after ingestion. Figure 1 (A-C) shows the scintiscans of 3 patients in whom discordant ¹⁴C-UBT results were obtained by employing the capsulated and non-capsulated protocols. For reference, the anterior contours of the stomach obtained 30 min after iv administration of 99mTcO4 - and before capsule ingestion are shown for each patient in order to exactly monitor the position and status of the ingested capsule. The clinical and ¹⁴C-UBT data of each patient is given below: Case No. 1: BNM, 45 years male, reported with symptoms of dyspepsia. Upper GI endoscopy and antral biopsy revealed hiatus hernia, esophagitis, antral hyperemia, multiple small ulcers in duodenum with presence of H. pylori infection. Scintigraphic image frame 3 of Figure 1A, shows that the capsule started leaking within 3 min of its ingestion and the tracer (¹⁴C-urea) moved out completely from the stomach within 7-8 min. Thus the scintigraphic images clearly demonstrate that there was total absence of ¹⁴C-urea in the stomach at the time when breath samples were collected at 10, 15 and 20 min. Hence, the capsulated ¹⁴C-UBT results did not show any presence of H. pylori infection. On the other hand, noncapsulated ¹⁴C-UBT results at 10 and 15 min were clearly indicative of H. pylori infection (Table 1).

Case No. 2: PK, 46 years male, reported with suspected dyspepsia. Upper GI endoscopy and antral biopsy revealed small gastric ulcer, antral gastritis with H. pylori infection. Scintigraphic image frame 2 of Figure 1B, shows that the capsule started leaking within 2 min of its ingestion and the tracer (¹⁴C-urea) almost moved out completely from the stomach within 3 min which subsequently entered into the intestine leaving the stomach completely free of tracer. The scintigraphic images demonstrate complete absence of ¹⁴C-urea in the stomach at 10 min. This resulted in negative ¹⁴C-UBT with capsulated UBT protocol; whereas, non-capsulated ¹⁴C-UBT results at all time points revealed the presence of H. pylori infection (Table 1).





Case No. 3: DS, 59 years male, reported with symptoms ofgastro-esophageal reflux disease. Upper GI endoscopy and antral biopsy revealed chronic active inflammation with H. pylori infection. Scintigraphic image frame 6 of Figure 1C shows that the capsule started leaking at sixth min and its contents started moving towards the duodenum. Subsequently, at the eighth min the entire capsule with its tracer moved out of the stomach leaving only trace amount of the ¹⁴C-urea in the stomach, which resulted in generation of low quantity of ¹⁴CO2 at all time points. Whereas, the non-capsulated ¹⁴C-UBT protocol showed exhalation of significant amount of ¹⁴CO2 especially at 10 and 15 min indicating the presence of H. pylori infection (Table 1).

Discussion

In this study, we report three H. pylori positive cases where we obtained discordant results of ¹⁴C-UBT by using the capsulated and non-capsulated protocols and found the noncapsulated method to be a superior option than the capsulated one for the diagnosis of H. pylori infection. This is the first study, which provides direct scintigraphy based evidence indicating that the misdiagnosis of H. pylori infection can occur mainly due to either rapid transit of ¹⁴C-urea containing capsule from the upper gastric tract or its incomplete resolution in the stomach during the phase of breath collection after ingestion. The capsulated ¹⁴C-UBT was introduced to avoid the problem of ¹⁴C-urea hydrolysis in the oropharynx due to presence of urease producing microorganisms and also to prevent the release of ¹⁴C-urea before it reaches the stomach.[8] These authors compared the results of capsulated and noncapsulated ¹⁴C-UBT protocols in a small number of patients without any consideration of their oral hygiene. They concluded that capsulated ¹⁴C-UBT protocol obviates the problem of false positive results in early breath samples and makes it possible to diagnose H. pylori infection with 99.8% reliability from a single 10 min breath sample. Further, Lerang et al[14] reported better specificity of capsulated ¹⁴C-UBT than the non-capsulated protocol (97% vs. 89%) with equal sensitivity for both (100%). While comparing both the ¹⁴CUBT protocols these authors did not keep the test conditions identical in terms of amount of tracer dose administered, timings of breath collection, expression of results etc. In addition, none of the above mentioned studies monitored the movement and fate of ¹⁴C-urea containing capsule after its ingestion. In the present study, while keeping all the test parameters identical we used a non-absorbable tracer marker (99mDTPA) along with ¹⁴C-urea in a capsule that helped us to delineate the exact position and fate of the capsule in the stomach while collecting breath samples for UBT simultaneously. In 2 of the present 3 discordant cases, the capsule with its contents moved out of the stomach before the first breath sample was obtained at 10 min; whereas in 1 case (DS) traces of ¹⁴C-urea leaked from the capsule in stomach which resulted in breath ¹⁴CO2 level below the cut-off value at all time points despite the fact that the patient had H. pylori infection. Thus there was no chance for ¹⁴C-urea present in the capsule to come in contact with the H. pylori infected site(s) in the stomach yielding false negative tests in these patients. On the other hand, employing the non-capsulated ¹⁴C-UBT protocol facilitated ¹⁴C-urea to immediately make contact with the entire stomach mucosa thereby initiating prompt hydrolysis of the urea by the H. pylori urease enzyme, as has been shown in Table 1.

Due to its high accuracy (>95%), the ¹⁴C-UBT assay provides a clinical “gold standard” against which the accuracy of other tests for the diagnosis of H. pylori infection can be validated.[9,14,20-23] Our preliminary observations showed that for the detection of active H. pylori infection, the use of liquid based non-capsulated ¹⁴C-UBT protocol appears to be a better option for the patients. However, it is important to validate these findings in a larger cohort of patients before we can draw tangible conclusions for patient management.

Our study concludes that the use of non-capsulated [14]CUBT appears to be a superior option over the conventional capsule based protocol for detection of H. pylori infection. Comparative data from a large number of patients needs to be generated further before one can arrive at definitive conclusions.

Declaration of funding

The study was supported by grants received from the Indian Council of Medical Research (ICMR), Government of India, vide Reference IRIS No. 2006-02600.

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