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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 55:M163-M167 (2000)
© 2000 The Gerontological Society of America

Noninvasive Diagnosis of Helicobacter pylori Infection in Older Subjects

Comparison of the 13C-Urea Breath Test With Serology

Alberto Pilottoa, Marilisa Franceschia, Gioacchino Leandrod, Mario Rassub, Rocco Maurizio Zagarie, Loredana Bozzolac, Francesca Furlanb, Franco Bazzolie, Francesco Di Mariof and Gianni Valerioa

a Department of Geriatrics, S. Bortolo Hospital, Vicenza, Italy.
b Microbiology Service, S. Bortolo Hospital, Vicenza, Italy.
c Clinical Pathology Service, S. Bortolo Hospital, Vicenza, Italy.
d Department of Gastroenterology, Castellana Grotte (BA), Italy.
e Department of Internal Medicine, University of Bologna, Italy
f Department of Gastroenterology, University of Padova, Italy.

Alberto Pilotto, Divisione Geriatria, Ospedale Civile S. Bortolo, Via Rodolfi, 37, 36100 Vicenza, Italy E-mail: alberto.pilotto{at}libero.it.

Decision Editor: William B. Ershler, MD


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Background. The potential influence of cognitive status, physical activities, comorbidity and cotreatments on the feasibility and diagnostic accuracy of two noninvasive diagnostic tests for Helicobacter pylori (Hp) infection, i.e., the 13C-urea breath test (13C-UBT) and serology (immunoglobulin G [IgG] anti-Hp antibodies), in older subjects is not known.

Method. The study involved 100 consecutive symptomatic elderly subjects (mean age, 78.3 years; range, 65–96 years), who had undergone an upper gastrointestinal endoscopy. Patients were considered Hp positive if at least two of the three invasive methods, i.e. histology, culture, and/or the rapid urease test were positive for Hp infection. Patients were considered Hp negative if all three invasive methods were negative. The 13C-UBT was performed according to the European standard method and the assaying of IgG anti-Hp antibodies by enzyme-linked immunosorbent assay. Cognitive status and functional activities were determined by the Mini-Mental State Examination (MMSE), the activities of daily living (ADLs) and instrumental ADLs (IADLs).

Results. According to invasive methods, 49 patients were Hp positive and 47 were Hp negative (4 subjects were excluded from the study). Hp-positive patients demonstrated a significantly higher prevalence of peptic ulcers (p=.02) and activity of chronic gastritis (p<.0001) than Hp-negative subjects. The 13C-UBT demonstrated a sensitivity of 100%, a specificity of 95.7%, and a diagnostic accuracy of 97.9%. Serology had significantly lower sensitivity (74.4%), specificity (59%), and diagnostic accuracy (67%, p<.001) than the 13C-UBT. The feasibility and the diagnostic accuracy of the 13C-UBT were not altered by the cognitive status (MMSE) and functional activities (ADL, IADL) of the patients, their drug consumption, or the prevalence of concomitant diseases.

Conclusions. In older subjects, the 13C-UBT had a significantly higher diagnostic accuracy than serology without influence of cognitive function, disability, comorbidity and cotreatments. This method may be considered an excellent, clinically useful, noninvasive test for the diagnosis of Hp infection in older subjects.

HELICOBACTER pylori (Hp) is currently considered the etiological agent of chronic gastritis type B, is strongly associated with peptic ulcer disease (1), and is linked to gastric atrophy and intestinal metaplasia (2), gastric cancer, and mucosa-associated lymphoid tissue lymphoma (3). The cure of Hp infection in elderly patients reduces symptomatology (4)(5), peptic ulcer recurrences (5), and the activity of chronic gastritis (6).

In the past decade, several methods have been developed for the diagnosis of Hp infection. These methods may be divided into two clinical groups: (i) invasive methods, i.e., histological evaluation, culture, the rapid-urease test, or polymerase chain reaction performed on gastric biopsies taken during upper gastrointestinal (GI) endoscopy and (ii) noninvasive methods, i.e., the measurement of immunoglobulin G (IgG) anti-Hp antibodies in serum or the 13C-urea breath test (13C-UBT [7]).

Few studies have compared the accuracy of these diagnostic methods in older subjects (8)(9), and no data have been published on the potential influence of the clinical characteristics of subjects on the feasibility and diagnostic accuracy of these diagnostic parameters.

The aims of the present study were: (i) to evaluate the sensitivity, specificity, and diagnostic accuracy of the 13C-UBT and IgG anti-Hp antibody serology in comparison with the invasive methods for Hp diagnosis in older patients and (ii) to evaluate the influence of cognitive function, disability, comorbidity, and cotreatments on the feasibility and diagnostic accuracy of these two noninvasive diagnostic methods.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Subjects
During a 3-month period, 100 consecutive subjects of 65 years of age or older who had undergone an upper GI endoscopy at the Digestive Pathophysiology Unit of the Geriatric Department in Vicenza, Italy, were included in the study. The inclusion criteria were the following: no treatment with antibiotics, proton pump inhibitors, or H2 blockers in the preceding four weeks; no previous anti-Hp treatments and/or upper GI surgery of any kind; and the availability of biopsy material during endoscopy. All patients had given informed consent for participation in the study.

Entry Assessment
Entry assessment included the collection of demographic data (age, gender), medication history, and current pathologies as documented by medical examination, clinical history, and clinical records from general practitioners. Evaluation of cognitive function was performed by means of the Mini-Mental State Examination (MMSE (10)). Physical function was assessed with standardized tests that evaluated the patient's ability to perform six activities of daily living (ADLs), i.e., bathing, dressing, transferring, walking, toilet functions, and eating (11) and seven instrumental ADLs (IADLs), i.e., managing finances, taking medications, using the telephone, shopping, using transportation, preparing meals, and doing housework (12).

Endoscopy and Biopsy Sampling
Macroscopic findings were recorded by one observer (A. Pilotto) and biopsies were taken from both the antrum, 5 cm proximal to the pylorus (four biopsies), and from the corpus, halfway along the greater curvature (four biopsies). Two antral and two corpus biopsies were analyzed histologically: one antral and one corpus biopsy were used for the rapid-urease test, and the remaining two (one from the antrum and one from the corpus) were used for the culture.

Histological Examination
Biopsy specimens were immediately fixed in buffered neutral formalin and embedded in paraffin. Sections were stained with hematoxylin–eosin and modified Giemsa for the detection of Hp. According to the Sydney system (13), chronic gastritis was defined histologically by the presence of chronic inflammatory cells in the lamina propria, and chronic gastritis activity was graded as none, mild, moderate or severe, according to the density of neutrophil granulocytes in the lamina propria, in intraepithelial sites, or both (13).

The Rapid-Urease Test
One antrum and one corpus biopsy specimen were used, with positivity assessed according to manufacturer's instructions (CLO test, Delta West Pty, Western Australia).

Microbiology
Gastric biopsy specimens taken during endoscopy (one from the antrum and one from the corpus) were placed in sterile tubes containing 1 ml of physiological saline and transported to the laboratory within 1 hour. Biopsies were cultured onto selective media (Helicobacter-selective agar plus 7% defibrinated horse blood, Becton Dickinson, Cockeysville, MD). The plates were incubated for 5–7 days at 37°C in microaerobic conditions (Campy Pak Plus, BBL, Becton Dickinson, Cockeysville, MD) and 100% relative humidity. Bacterial growth was identified as Hp on the basis of Gram stain, colony morphology, and biochemical properties (14).

IgG Anti-Hp Antibodies
A blood sample was collected from all subjects for the determination of serum IgG anti-Hp antibodies using an established enzyme-linked immunosorbent assay (Enzygnost Anti-Helicobacter pylori II/IgG, Dade Behring, Marburg, Germany) (15). The levels of specific IgG anti-Hp antibodies were derived from a reference-positive sample by means of photometric evaluation of optical density at 450 nm. The results were expressed in units per milliliter: a cutoff point of greater than 10 units/ml indicated positivity. The manufacturer reported a sensitivity and specificity of the test of 93.1% and 98%, respectively.

13C-Urea Breath Test
The 13C-UBT was performed according to the European Standard Protocol (16). Fasting patients were given a fatty test meal (60% lipid, 25% carbohydrate, 15% protein; 100 ml, Pulmocare, Abbott Laboratories) to delay gastric emptying. A baseline breath sample was then collected with a disposable plastic straw. After 10 minutes, patients drank 100 mg of 13C-urea (99%, Tracer Technologies Inc., Somerville, MA) dissolved in 50 ml of water. A single-point breath sample was taken after 30 minutes. All samples were tested twice. The ratio of 13C-urea to 12C-urea in expired breath was measured by mass spectrometry and expressed as 13CO2 (per milliliter). An automated breath 13C analyzer was used; excess 13CO2 excretion greater than 5% was considered positive for Hp infection (17).

Definition of Hp Infection
Patients were considered to be infected with Hp if two or more invasive methods, i.e., histology, culture, and/or the rapid-urease test, were positive for Hp infection. Patients were considered not infected if all three invasive methods were negative for Hp infection. Any subjects who presented Hp positivity by histology but not by the rapid-urease test or culture were considered undefined and were excluded from the study.

Statistics
Sensitivity, specificity, and diagnostic accuracy were calculated according to standard methods. Differences between Hp-positive and Hp-negative subjects with regards to cognitive and physical functions, cotreatments, comorbidity, and endoscopic diagnoses were evaluated by means of the Student's t test for unpaired data and the Mann–Whitney test. Differences in diagnostic accuracy between serology and the 13C-UBT were evaluated by means of the chi-square analysis. The Biomedical Data Processing package (BMDP, University of California, Los Angeles, CA) was used with the Biomedical Data Processing default parameters. All p values were two tailed with statistical significance indicated by a value of p < .05.


   Results
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 Abstract
 Methods
 Results
Discussion
 References
 
Table 1 illustrates the epidemiological and clinical characteristics of the 49 Hp-positive (18 men, 31 women; mean age, 77.9 years; range: 65–96 years) and 47 Hp-negative subjects (19 men, 28 women; mean age, 77.3 years; range: 65–90 years). No differences were found between Hp-positive and Hp-negative patients as regards MMSE, ADL, and IADL scores as well as drug consumption and concomitant extradigestive diseases. Hp-positive subjects demonstrated a significantly higher prevalence of peptic ulcer disease (p=.02) and of moderate–severe activity of chronic gastritis (p<.0001) than Hp-negative subjects (Table 2 ).


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  		Table 1. Characteristics of Patients Stratified According to the Presence of Hp Infection

 

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  		Table 2. Upper GI Diagnoses as Documented at Endoscopy in Patients Stratified According to Hp Infection

 
As reported in Table 3 , the 13C-UBT demonstrated a sensitivity of 100% and a specificity of 95.7% (two patients resulted false positive). Sensitivity and specificity of IgG anti-Hp antibody assay were respectively 74.4% (12 patients were false negative) and 59% (18 subjects were false positive). Thus the diagnostic accuracy of 13C-UBT was significantly higher than the IgG anti-Hp antibody assay (97.9% versus 67%, p<.001).


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  		Table 3. Sensitivity, Specificity, and Diagnostic Accuracy of the 13C-UBT Versus the Assaying of IgG anti-Hp Antibodies in Serum in Older Subjects

 
No differences in mean age, MMSE, ADL, IADL, drug consumption, or the prevalence of concomitant diseases were found among the 61 subjects who were correctly diagnosed by serology (true-positive and true-negative subjects) versus the 30 false-positive and false-negative subjects. False-negative patients did demonstrate a trend toward a higher mean age, lower values of MMSE, ADL, and IADL scores, and higher drug consumption (Table 4 ).


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  		Table 4. Characteristics of Patients Correctly Diagnosed by Serum Assaying of IgG Anti-Hp Antibodies (True-Positive and True-Negative) Compared with False-Positive and False-Negative Subjects

 
The two patients who resulted false positive by the 13C-UBT were a 72-year-old man in good clinical condition with regards to cognitive and physical function and a 93- year-old woman with comparatively lower MMSE, ADL, and IADL scores.


   Discussion
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 Abstract
 Methods
 Results
 Discussion
References
 
The diagnosis of Hp infection has become a standard procedure in the management of patients with upper gastrointestinal pathology. Recently the European Helicobacter Pylori Study Group advised that Hp diagnosis should occur at the general practitioner level (18). As a consequence, noninvasive diagnostic tests will become increasingly important in clinical practice.

Very few studies have explored the clinical feasibility and diagnostic accuracy of noninvasive methods for the diagnosis of Hp infection in older subjects (8)(9)(19), particularly regarding the potential for a patient's cognitive and physical status to affect test results.

This study was performed in 100 consecutive older subjects who had undergone an upper GI endoscopy as either outpatients (54 subjects) or inpatients (46 subjects). The mean scores of the cognitive and the functional activity tests, i.e., MMSE, ADL, and IADL, were relatively high in this elderly population; however, the wide range of data (from 9 to 30 for MMSE, from 1 to 12 for ADL, and from 0 to 14 for IADL) indicated that the study population was a very heterogenous group with a wide range of different clinical characteristics and resultant cognitive and physical abilities.

The study demonstrated that in older subjects the 13C-UBT had excellent sensitivity, specificity, and diagnostic accuracy (97.9%). In contrast, serology showed low sensitivity (12 false negatives of 47) and specificity (18 false positives of 44) with a diagnostic accuracy (67%) significantly lower than that of the 13C-UBT. These data are in agreement with the results of previous studies indicating in older patients a significantly different prevalence of Hp infection when detected by serology versus histology (20) or the 13C-UBT (21).

The lack of accuracy and sensitivity of the serum anti-Hp assay confirms that this should not be the recommended diagnostic test in elderly subjects because it does not accurately reflect the older patient's true gastric Hp status (21). Furthermore, poor serum results appear to be particularly associated with the older population. A recent study (22) that compared seven methods for the diagnosis of Hp infection in 97 subjects with a mean age of 58 years for females and 53 years for males (range 18 to 85 years), reported notably higher serology results: 81% sensitivity, 78% specificity, and 79% accuracy, than the presently reported 74.4%, 59%, and 67%. The cited study also confirmed in a heterogenous population the higher accuracy (87%), sensitivity (90%), and specificity (89%) of 13C-UBT results.

There are several possible explanations for the poor results of serology in the elderly. False positivity may be due to the increased prevalence of atrophic gastritis that occurs with age (2). Under these local gastric conditions, Hp colonization may disappear whereas specific IgG anti-Hp antibodies are known to linger in circulation (19). An explanation for the absence of serum anti-Hp antibodies in gastric Hp-positive patients is less forthcoming. A possible reason could be the immune incompetence of older patients, as suggested by James and colleagues (23). In the present study, however, no differences were observed in the prevalence of concomitant diseases, particularly immunological diseases, between the patients correctly diagnosed by serology in comparison with false-negative subjects. Indeed, the latter group of subjects did have a trend toward a higher age, lower cognitive and functional skills, and a higher drug consumption.

The diagnostic accuracy of the 13C-UBT was not influenced by the presence of concomitant diseases and therapies (excluding protein pump inhibitors and antibiotics). The test was easily performed in subjects with relatively low MMSE, ADL, and IADL scores, indicating that minimal collaboration by the patients is needed for the correct performance of the test.

In both Hp-positive and Hp-negative subjects, a high prevalence of organic lesions, including esophagitis, erosive gastritis, gastric or duodenal ulcers, as well as esophageal and gastric cancers, was documented at endoscopy. These data confirm the general clinical opinion that in older symptomatic patients, endoscopy must be the first line diagnostic approach. Noninvasive methods for the detection of Hp infection should be reserved for older subjects who cannot undergo an endoscopy, for patients in whom it is not possible to take gastric biopsies during endoscopy (i.e., in cases of coagulation impairment), or as a marker for the efficacy of anti-Hp eradication therapy. Recently, new noninvasive diagnostic tests have been developed involving the detection of antibodies in saliva and full blood (24) or Hp antigens in feces (25). These methods, however, have not yet been evaluated sufficiently to confirm their reliability and potential role in the diagnosis of Hp infection (7), particularly in older populations.

In conclusion, in older subjects the 13C-UBT demonstrated a significantly higher diagnostic accuracy than the assaying of IgG anti-Hp antibodies in serum. Parameters of cognitive status (MMSE) and functional activities (ADL, IADL), comorbidity, and cotreatments did not influence the feasibility and diagnostic accuracy of the 13C-UBT. This method may be considered an excellent, clinically useful noninvasive test for the diagnosis of Hp infection in older subjects who present with a wide range of cognitive and physical characteristics.

Received March 17, 1999

Accepted August 18, 1999


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

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