- Email: [email protected]
Accurate Diagnosis of Helicobacter pylori
HELICOBACTER PYLORI, PART I1
0889-8553/00 $15.00
+ .OO
ACCURATE DIAGNOSIS OF HELICOBACTEX PYLORI Stool Tests Din0 Vaira, MD, Marcello Menegatti, MD, Chiara Ricci, MD, Luigi Gatta, MD, Sonia Berardi, MD, and Mario Miglioli, MD
Helicobacter pylori is a human pathogen that causes chronic gastritis, has a role in gastric and duodenal ulcer, is involved in gastric carcinogenesis (so that the bacterium has been classified as a class I definite gastric carcinogen to human^),^ and is regarded as a possible important factor in at least a subset of patients with functional dyspepsia. In addition to its definite role as a gastroduodenal pathogen, H. pylori now is being investigated actively for possible involvement in various nongastroenterologic conditions, such as impaired growth, coronary heart disease, headache, Raynaud's phenomenon, diabetes, and gallstone disease? H. pylori causes a chronic gastric infection that usually is lifelong, and many epidemiologic studies have shown that this is probably one of the most common bacterial infections throughout the world, involving 40% to 50% of the population in developed countries and 80% to 90% of the population in developing regions.z1The diagnosis of H. pylori infection represents at least a key step in the management of many patients referred to the gastroenterologist. Because of the wide range and relevance of pathologies possibly related to infection (including malignancies), H. pylori harbors the potential to become a major health problem. H. pylori infection can be diagnosed by invasive (i.e., requiring endoscopy) and noninvasive techniques (i.e., techniques that do not require endoscopy with biopsy sampling)." Each of the available diagnostic techniques (which are discussed in detail in other articles) has advantages and disadvantages. The discussion of the different diagnostic methods cannot be oversimplified by
From the Department of Internal Medicine, University of Bologna, Bologna, Italy
GASTROENTEROLOGY CLINICS OF NORTH AMERICA
-
VOLUME 29 NUMBER 4 * DECEMBER 2000
917
918
VAIRAetal
thinking only in terms of which is the best diagnostic tool. The problem should be addressed by asking which is the best diagnostic tool in each definite clinical situation. The choice of diagnostic tool has to take into account different factors, as follows: Is the clinician dealing with normal subjects screened for epidemiologic purposes or with patients referred to the gastroenterologist?Has the patient already had failed eradication attempts? Is the clinician looking for susceptibility to antibodies? Is the clinician interested in diagnosing the infection in a clinical setting or in other possibly relevant factors (i.e., putative markers of increased virulence or pathogenicity of the strain, such as cugA or vucA) in a research setting? Eventually the clinician also should ask the cost of the diagnostic technique used, taking into account all the factors involved, such as the need for endoscopy, a technician or nurse to assist the patient, and dedicated laboratory instrumentation and materials (i.e., to evaluate breath sample) as well as available facilities (in the case of serologic tests, facilities usually are available even in small hospitals or developing countries). Bearing in mind these and other similar questions, the possibility of testing stool samples to diagnose H. pylori infection noninvasively is discussed. Previously, there were only two widely available noninvasive methods: (1)13C-labeledor 14C-labeledurea breath test, which is based on the detection of I3C-labeled or 14C-labeledcarbon dioxide expired air as result of H. pylori urease activity: l1 and (2) serologic testing, which is based on the detection of a specific .anti-H. pylori immune response, mostly by IgG antibodies, in the patienYs serum.", 22 Being noninvasive, these tests (mostly serologic testing because of its simplicity and lower cost) have been used widely in epidemiologic studies to assess the prevalence of H. pylori infection in different populations. Apart from epidemiologic research, noninvasive H. pylori testing can be used successfully in two other main settings: (1) pre-endoscopic screening of patients to refer to a gastroenterology service for investigation of dyspepsia and (2) therapeutic monitoring after eradication therapy to confirm cure of infection. Because of the widespread availability of endoscopy, this technique has become the mainstay for investigation of dyspepsia, and this has led to increased waiting lists and medical costs. In an attempt to obviate the need for endoscopy, without affecting the safety of the patients, several pre-endoscopic screening procedures have been proposed, and it has been shown that young H. pylori-negative patients (as determined by noninvasive tests) without alarming symptoms or nonsteroidal anti-inflammatory drug intake could avoid endoscopy safely and be treated with a trial of medical therapy. Endoscopy could be reserved for patients whose symptoms do not improve or relapse shortly after discontinuation of therapy.I8 Apart from patients with gastric ulcer, who probably are managed best with control endoscopy because of the possibility of underlying gastric malignancy, two approaches have been advocated for patients treated with H. pylorieradicating regimens. The first approach is to wait and see (i.e., not testing to confirm eradication, suggesting that if patients experience prolonged symptom relief, this could be taken as an indirect sign of successful eradication). This approach may not be accepted by some patients, for whom fear of consequences of H. pylori infection dictates a positive confirmation of eradication. Such a strategy is based on the unproven assumption of a clear relationship between H. pylori and symptoms. The second approach is to test and confirm eradication, and this probably is best accomplished by noninvasive testing. Urea breath test can be used 4 weeks after treatment, whereas serologic testing requires waiting a longer time to allow the antibody titer to fall.
ACCURATE DIAGNOSIS OF HELICOBACTER PYLORI
919
HELICOBACTER PYLORI STOOL ASSAYS
Culture of H. pylori has been obtained from stool samples, but viable organisms are present only in a small percentage of cases.17Despite the difficulties encountered in culture from stool samples, the fact that the organisms were present at all raised the possibility of developing a new noninvasive diagnostic test based on the detection of bacterial antigen in stool. An enzymatic immunoassay that detects the presence of H. pylori antigen in stool specimen has become available (HpSA, Meridian Diagnostics, Cincinnati, OH) and has begun to be tested in clinical practice to evaluate its performance compared with that of the other already available diagnostic tests. The HpSA test has received approval from the US Food and Drug Administration for two indications: (1) diagnosis of H. pylori infection in symptomatic adults and (2) monitoring response and posttherapy in adults. The test uses polyclonal anti-H. pylori capture antibody absorbed to microwells. The stool specimen can be stored at 2°C to 8°C for 3 days or indefinitely at -20°C before the test. This storage makes possible the collection of multiple samples over several days to weeks (important mostly in small hospitals with a small number of patients) to be tested in the same session, reducing the cost, and it allows the storage of samples that could be used in future analysis. A small portion of the specimen is diluted with a sample diluent, and no further manipulation is needed. Diluted fecal samples and a peroxidase conjugated polyclonal antibody are added to the wells and incubated for 1 hour at room temperature, then a washing step is performed to remove unbound material. Substrate is added and incubated for 10 minutes at room temperature. In the presence of bound H . pylori antigens, a color develops. Finally, a stop solution is added, and the results are read spectrophotometrically (450 nm). According to the manufacturer's instruction, the cutoff values are as follows: less than 0.140, negative (i.e., no H. pylori antigens in the stool samples, patients not infected); 0.140 to 0.159, equivocal-indeterminate (according to the manufacturer's instruction, indeterminate results should be repeated; if the repeated result is equivocal, a new specimen should be obtained); and greater than or equal to 0.160, positive (i.e., H. pylori antigens are present in the stool samples, patients infected). Such a test, which detects bacterial antigen in an ongoing infection, theoretically is useful not only for screening, but also as an early predictor of successful treatment. The authors consider briefly the currently available evidence supporting a possible role for this noninvasive diagnostic test. There are relatively few studies evaluating HpSA, most of which evaluate limited populations, assessing diagnostic accuracy in terms of sensitivity and specificity compared with more standardized techniques, and this is even more the case for assessing patients after eradication. An exception is a European multicenter study carried out in 11 endoscopic units with a specific interest in H. pylori infection throughout E u r ~ p e . 'The ~ first part of this prospective study was planned to assess the accuracy of HpSA compared with standardized technique; the authors enrolled 501 consecutive patients (not previously treated for H. pylori infection), who underwent endoscopy with multiple biopsies (for histology in antrum and corpus, rapid urease test, and culture) and I3C-urea breath test. A stool sample was collected from all patients. According to guidelines for clinical trialP in H. pylori infection, patients were considered H. pylori positive if at least two tests were positive; if culture alone was positive, in view of its absolute specificity, the patient also was considered positive. Using this gold standard, the overall sensitivity and specificity of the two noninvasive tests were 94% (confidence
920
VAIRAetal
Table 1. PROSPECTIVE EUROPEAN MULTICENTER STUDY SENSITIVITY AND SPECIFICITY OF HELlCOBACTER PYLORI STOOL ANTIGEN COMPARED WITH 13C-UREA BREATH TEST BEFORE AND AFTER TREATMENT ~~
~~~
Before Treatment (n = 501)
Sensitivity (“h) Specificity (%) HpSA
=
After Treatment (n= 133)
HpSA
“C-UBT
HpSA
‘3C-UBT
94.3 91.8
95.3 97.7
92.3 96.2
88.5 99.4
H.pylori stool antigen; W-UBT
=
’jC-urea breath test.
interval [CI], 90.6 to 96.6) and 91.8% (CI, 87.3 to 95.1) versus 95.3% (CI, 92.2 to 97.5) and 97.7% (CI, 94.8 to 99.3) for HpSA and 13C-ureabreath test (Table 1).In this study, a clear-cut result was obtained for most patients, with only 10 of 501 (2%) equivocal results. Of the 501 patients enrolled, 279 were found to be H. pylori positive, were given an eradicating treatment, and were asked to return 4 weeks after therapy for follow-up endoscopy, 13C-urea breath test, and HpSA. The authors have reported on 206 patients followed up: 133 by endoscopy, 13C-urea breath test, and HpSA and 73 by 13C-ureabreath test and HpSA only.2oIn the 133 patients (a population numerically important for a follow-up study) who underwent endoscopy, the overall sensitivity and specificity of HpSA and 13C-urea breath test were 92.3% and 96.2% versus 88.5% and 99.1% (see Table 1). The test gave clear-cut results in most patients: 132 of 133 (99.2%). Most of the remaining clinical studies on this test so far have been only as abstracts? In most cases, these studies confirm the excellent performance of the HpSA before and after therapy (although most studies involved limited populations); a few reports obtained considerably lower performance in terms of sensitivity and specificity, highlighting the need for further large validation studies (Table 2). In addition to evaluating the accuracy of the HpSA test compared with other techniques, other issues are being addressed by investigators throughout the world, such as the possible influence of concomitant use of proton-pump inhibitors (PPIs),” the kinetics of stool antigen during and after therapy,13,25 the possibility of cross-reaction with antigen from bacteria different from H. pylori,16 the possible use in pediatric populations,” the possibility that HpSA may detect rapid urease test-negative and histology-negative H. pylori-infected patients? and the cost-effectiveness of the test.= Vakil et alZ4evaluated the possible effect of concomitant use of PPIs on stool antigen secretion and found no differences between the value observed in 10 patients receiving PPIs (0.55k0.32) and 45 patients not receiving PPIs (0.61720.135). van’t Hoff et alZ5used the HpSA test to monitor the disappearance of H. pylori infection in the stomach during and shortly after eradication in a subgroup of 19 patients enrolled in the previously mentioned European multicenter study. The authors showed a fast decline of H. pylori antigen levels in the first 3 days after the start of the therapy, with all but two patients being below the cutoff value at 5 days. These results led the authors to conclude that if these data are confirmed by further studies, detection of HpSA would change dramatically the management of H. pylori patients. The same topic was addressed by Petersen et all3 who investigated 20 H. pyloripositive patients who were asked to provide a stool sample before initiating “References 1-3, 5, 8, 10, 12-14, 16, 24-26.
921
ACCURATE DIAGNOSIS OF HELICOBACTER PYLORI
Table 2. SENSITIVITY AND SPECIFICITY OF HELlCOBACTER PYLORl STOOL ANTIGEN’ Before Treatment (n) Author
Braden et all Costa et a12 Feliciangeli et a13 Monteiro et all2 vakii24 Wirtheim et alZ6 Grubel et a15 Kim et a18 Lujan et all0 Sander et all4
After Treatment (n)
Sensitivity
Specificity
Sensitivity
Specificity
(W
@Y
(W
(W
95 (54) 94 (124) 80 (91) 87 (97) 90 (41) 96 (73) 100 (19) 100 (41)
-
-
90 (45)
80 (45)
-
-
93 97 58.8 88.4 87 83 88 87.4 97.3 98
(54) (124) (91) (97) (41) (73) (19) (41) (38) (137)
-
95 (137)
-
-
100 (14)
100 (14)
-
-
-
95 (26)
100 (26)
-
-
*Abstracts presented at the Digestive Disease Week (DDW), 1999 (see individual references).
eradicating therapy, then every other day for 2 weeks. In the 17 patients who completed the study, HpSA levels in stool usually increased, with a peak seen between 2 and 5 days, after which it decreased to undetectable levels if the treatment was successful. This peak in HpSA levels in the first 2 to 5 days of therapy was considered to be owing to the bactericidal effect of the antibioticreleasing antigen from the killed bacteria. The authors found that if the treatment was unsuccessful, the HpSA increased to approximately the same levels as seen initially over a few days. Taylor et all6evaluated the HpSA for possible cross-reactivateswith Helicobacter species other than H. pylori (including H . canis, H . rodentium, H . felis, H . pullorurn, H. cineady, H. fenneliae, H. bilis, H. hepaticus, H. rappini, and H . bizzozzeroni). The study was carried out in animal models using feces from Helicobacterinfected mice, ferrets, and cats. The authors found that other Helicobacter species, some of which have been reported in human disease, react in the H . pylori antigen capture assay and concluded that these data require confirmation in human studies to determinate the relevance of these findings in a clinical setting. Scant data are available on pediatric populations; however, at least two studies6, have included children. Both of these studies found a good performance of the stool test, and Kim et a18 concluded that the technique may be a substitute for endoscopy, especially in children and some patients unable to undergo endoscopic examination. Hsu et a16investigated four H. pylori-positive patients undergoing eradication treatment. In these patients, stool samples were collected every other day during antibiotic treatment and at 4 weeks after treatment, and the eradication was confirmed by 13C-urea breath test, biopsy specimens from antrum and corpus for culture, CLO test, and histology. Hsu et a16 found that at 4 weeks incomplete eradication in two patients resulted in positive urea breath test and HpSA but negative endoscopy tests, and they concluded that larger scale studies are needed to verify the cost-effectiveness of HpSA as a noninvasive test for eradication. The topic of cost-effectiveness of HpSA was addressed by Vakil,= who modeled a study on the cost-effectiveness of stool testing, urea breath test, office serology, enzyme-linked immunosorbent assay serology, and whole-blood testing in the initial diagnosis of the H . pylori infection and the cost-effectiveness
922
VAIRAetal
of urea breath test and stool studies in patients given eradication therapy. In this mathematical model, VakiP found that before therapy the stool test compared favorably with the other technique assuming a prevalence of H. pylori infection of 30% in dyspeptic patients, and when the prevalence of infection dropped to 19% the stool test was the most cost-effective because of its higher specificity than office-based blood or serum tests. In posttherapy testing, the stool test was cost-effective compared with 14C-ureaand 13C-ureabreath test with an average cost of $183 2 197 versus 213 5 9 9 versus 369 5 105. The conclusions were that (1) stool tests are cost-effective alternatives to office-based serology testing, and (2) their most promising role is in posttreatment assessment. SUMMARY
From the limited data available, it seems that the H. pylori stool assay represents a highly accurate diagnostic tool to detect H. pylori infection before and shortly after therapy. As a test that is noninvasive, accurate, simple, and cost-effective, the H. pylori stool assay has the potential to become the preferred diagnostic tool in many different clinical settings from epidemiologic studies to pediatric investigations, from pre-endoscopic screening policies to posttherapy monitoring. References 1. Braden B, Dietrich CF, Teuber G, et al: New antigen test in stool detects Helicobacter pylori infection: Non invasive diagnostic tool for therapy control. Gastroenterology 116A127, 1999 2. Costa F, Bellini M, Mummolo G, et al: A new enzyme immunoassay to detect Helicobacter pylori (HI') antigens on stool specimens: Diagnostic accuracy before and after eradicating treatment. Gastroenterology 116:A139, 1999 3. Feliciangeli G, Macarri G, Di Sario A, et al: Diagnostic accuracy of PCR in the detection of Helicobacter pylori infection. Gastroenterology 116:A160, 1999 4. Gasbarrini A, Franceschi F, Gasbarrini G, et al: Extraintestinal pathology associated with Helicobacter infection. Eur J Gastroenterol Hepatol9:231-233, 1997 5. Grubel P, Franck RW, Willis DH, et ak HpSA enzyme immunoassay for the detection of H pylori from stool samples collected from a field in Egypt. Gastroenterology 116A177, 1999 6. Hsu RK, Solnick J, Ruebner B: Premier HpSA Helicobacter pylori stool antigen may detect CLO and histology negative infection after antibiotic therapy: A prospective study. Gastroenterology 116A191, 1999 7. International Agency for Research on Cancer, World Health Organisation: Infection with Helicobacter pylori. In: Schistosomes, Liver Flukes and Helicobacter pylori. Lyon, IARC, 1994, pp 177-202 8. Kim PS, Lee J, Pai SH, et al: Detection of Helicobacter pylori antigen in stoolmby ELISA. Gastroenterology 116:A215, 1999 9. Logan RPH: The urea breath test. In Lee A, Mkgraud F (eds): Helicobacter pylori: Technique for Clinical Diagnosis and Basic Research. London, WB Saunders, 1996, PP 74-81 10. Lujan M, I'amos S, Llucian R, et al: Sensitivity and specificity of H pylori antigen in faeces. Gastroenterology 116:A245, 1999 11. Megraud F How should Helicobacter pylori infection be diagnosed? Gastroenterology 112:S9>S98, 1997 12. Monteiro L, De Mascarel A, Barberis C, et al: What is the performance of the Premier Platinum HpSA test in comparison to other invasive tests for the detection of Helicobacter pylori infection? Gastroenterology 116A258, 1999
ACCURATE DIAGNOSIS OF HELICOBACTER PYLORI
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13. Petersen JR, Adesoji 8, Okorodudu A, et al: Using the Helicobacter pylori stool antigen (HpSA) in monitoring antibiotic treatment response of Helicobacter pylori. Gastroenterology 116A281, 1999 14. Sander JO, van Zanten V, Bleau BL, et al: Helicobacter pylori stool antigen test (HpSA): Use for detection of infection and eradication. Gastroenterology 116:A3639, 1999 15. Technical annex: Tests used to assess Helicobacter pylori infection: Guidelines for clinical trials in Helicobacter infection. Working party of the European Helicobacter pylori Study Group. Gut 4l(suppl2):Sl(rS18, 1997 16. Taylor NS, Esteves M, Fox JG: Cross reactivity with Helicobacter species assayed with a Helicobacter pylori antigen capture assay. Gastroenterology 116:A830, 1999 17. Thomas JE, Gibson GR, Darboe MK, et al: Isolation of Helicobacter pylori from human faeces. Lancet 340:1194-1195, 1992 18. Vaira D, et al: Prospective screening of dyspeptic patients by Helicobacter pylori serology: A safe policy? Endoscopy 29:595-601, 1997 19. Vaira D, Malfertheiner P, MCgraud F, et al, and European Helicobacter pylori HPSA Study Group: Diagnosis of Helicobacter pylori infection using a novel, noninvasive antigen based assay in a European multicentre study. Lancet 354:30-33, 1999 20. Vaira D, Malfertheiner P, MCgraud P, et al, and European HpSA Study Group: Non invasive tests for monitoring Helicobacter pylori (HP). European Multicentre Study. Gastroenterology 116:A341, 1999 21. Vaira D, Miglioli M, Mule P, et al: Prevalence of peptic ulcer in Helicobacter pylori positive blood donors. Gut 35:309-312, 1994 22. Vaira D, Stanghellini V, Menegatti M, et al, and The Italian Helicobacter Study Group: IgG ELISA antibodies and detection of Helicobacter pylori in elderly patients. Lancet 1~269-270,1996 23. Vakil N: Cost effectiveness of non invasive testing methods for H pylori before in dyspeptic patients. Gastroenterology 116:AlOO, 1999 24. Vakil N, Affi A, Sundaram M, et al: Prospective blinded evaluation of the accuracy of a stool test for the detection of H pylori. Gastroenterology 116:A342, 1999 25. van’t Hoff BMW, Vaira D, van der Ende A, et al: A novel non invasuve test to assess Helicobacter pylori (Hp) shortly after eradicating treatment. Gastroenterology 116:A341, 1999 26. Wirtheim E, Yahav J, Mauz E, et al: The sensitivity and specificity of a novel enzyme immunoassay (Meridian HpSA) for the detection of Helicobacter pylori antigen stool specimens. Gastroenterology 116:A355, 1999
Address reprint requests to Din0 Vaira, MD Clinica Medica I Universiti di Bologna Policlinico S. Orsola V. Massarenti g 40138 Bologna Italy e-mail: [email protected]
0889-8553/00 $15.00
+ .OO
ACCURATE DIAGNOSIS OF HELICOBACTEX PYLORI Stool Tests Din0 Vaira, MD, Marcello Menegatti, MD, Chiara Ricci, MD, Luigi Gatta, MD, Sonia Berardi, MD, and Mario Miglioli, MD
Helicobacter pylori is a human pathogen that causes chronic gastritis, has a role in gastric and duodenal ulcer, is involved in gastric carcinogenesis (so that the bacterium has been classified as a class I definite gastric carcinogen to human^),^ and is regarded as a possible important factor in at least a subset of patients with functional dyspepsia. In addition to its definite role as a gastroduodenal pathogen, H. pylori now is being investigated actively for possible involvement in various nongastroenterologic conditions, such as impaired growth, coronary heart disease, headache, Raynaud's phenomenon, diabetes, and gallstone disease? H. pylori causes a chronic gastric infection that usually is lifelong, and many epidemiologic studies have shown that this is probably one of the most common bacterial infections throughout the world, involving 40% to 50% of the population in developed countries and 80% to 90% of the population in developing regions.z1The diagnosis of H. pylori infection represents at least a key step in the management of many patients referred to the gastroenterologist. Because of the wide range and relevance of pathologies possibly related to infection (including malignancies), H. pylori harbors the potential to become a major health problem. H. pylori infection can be diagnosed by invasive (i.e., requiring endoscopy) and noninvasive techniques (i.e., techniques that do not require endoscopy with biopsy sampling)." Each of the available diagnostic techniques (which are discussed in detail in other articles) has advantages and disadvantages. The discussion of the different diagnostic methods cannot be oversimplified by
From the Department of Internal Medicine, University of Bologna, Bologna, Italy
GASTROENTEROLOGY CLINICS OF NORTH AMERICA
-
VOLUME 29 NUMBER 4 * DECEMBER 2000
917
918
VAIRAetal
thinking only in terms of which is the best diagnostic tool. The problem should be addressed by asking which is the best diagnostic tool in each definite clinical situation. The choice of diagnostic tool has to take into account different factors, as follows: Is the clinician dealing with normal subjects screened for epidemiologic purposes or with patients referred to the gastroenterologist?Has the patient already had failed eradication attempts? Is the clinician looking for susceptibility to antibodies? Is the clinician interested in diagnosing the infection in a clinical setting or in other possibly relevant factors (i.e., putative markers of increased virulence or pathogenicity of the strain, such as cugA or vucA) in a research setting? Eventually the clinician also should ask the cost of the diagnostic technique used, taking into account all the factors involved, such as the need for endoscopy, a technician or nurse to assist the patient, and dedicated laboratory instrumentation and materials (i.e., to evaluate breath sample) as well as available facilities (in the case of serologic tests, facilities usually are available even in small hospitals or developing countries). Bearing in mind these and other similar questions, the possibility of testing stool samples to diagnose H. pylori infection noninvasively is discussed. Previously, there were only two widely available noninvasive methods: (1)13C-labeledor 14C-labeledurea breath test, which is based on the detection of I3C-labeled or 14C-labeledcarbon dioxide expired air as result of H. pylori urease activity: l1 and (2) serologic testing, which is based on the detection of a specific .anti-H. pylori immune response, mostly by IgG antibodies, in the patienYs serum.", 22 Being noninvasive, these tests (mostly serologic testing because of its simplicity and lower cost) have been used widely in epidemiologic studies to assess the prevalence of H. pylori infection in different populations. Apart from epidemiologic research, noninvasive H. pylori testing can be used successfully in two other main settings: (1) pre-endoscopic screening of patients to refer to a gastroenterology service for investigation of dyspepsia and (2) therapeutic monitoring after eradication therapy to confirm cure of infection. Because of the widespread availability of endoscopy, this technique has become the mainstay for investigation of dyspepsia, and this has led to increased waiting lists and medical costs. In an attempt to obviate the need for endoscopy, without affecting the safety of the patients, several pre-endoscopic screening procedures have been proposed, and it has been shown that young H. pylori-negative patients (as determined by noninvasive tests) without alarming symptoms or nonsteroidal anti-inflammatory drug intake could avoid endoscopy safely and be treated with a trial of medical therapy. Endoscopy could be reserved for patients whose symptoms do not improve or relapse shortly after discontinuation of therapy.I8 Apart from patients with gastric ulcer, who probably are managed best with control endoscopy because of the possibility of underlying gastric malignancy, two approaches have been advocated for patients treated with H. pylorieradicating regimens. The first approach is to wait and see (i.e., not testing to confirm eradication, suggesting that if patients experience prolonged symptom relief, this could be taken as an indirect sign of successful eradication). This approach may not be accepted by some patients, for whom fear of consequences of H. pylori infection dictates a positive confirmation of eradication. Such a strategy is based on the unproven assumption of a clear relationship between H. pylori and symptoms. The second approach is to test and confirm eradication, and this probably is best accomplished by noninvasive testing. Urea breath test can be used 4 weeks after treatment, whereas serologic testing requires waiting a longer time to allow the antibody titer to fall.
ACCURATE DIAGNOSIS OF HELICOBACTER PYLORI
919
HELICOBACTER PYLORI STOOL ASSAYS
Culture of H. pylori has been obtained from stool samples, but viable organisms are present only in a small percentage of cases.17Despite the difficulties encountered in culture from stool samples, the fact that the organisms were present at all raised the possibility of developing a new noninvasive diagnostic test based on the detection of bacterial antigen in stool. An enzymatic immunoassay that detects the presence of H. pylori antigen in stool specimen has become available (HpSA, Meridian Diagnostics, Cincinnati, OH) and has begun to be tested in clinical practice to evaluate its performance compared with that of the other already available diagnostic tests. The HpSA test has received approval from the US Food and Drug Administration for two indications: (1) diagnosis of H. pylori infection in symptomatic adults and (2) monitoring response and posttherapy in adults. The test uses polyclonal anti-H. pylori capture antibody absorbed to microwells. The stool specimen can be stored at 2°C to 8°C for 3 days or indefinitely at -20°C before the test. This storage makes possible the collection of multiple samples over several days to weeks (important mostly in small hospitals with a small number of patients) to be tested in the same session, reducing the cost, and it allows the storage of samples that could be used in future analysis. A small portion of the specimen is diluted with a sample diluent, and no further manipulation is needed. Diluted fecal samples and a peroxidase conjugated polyclonal antibody are added to the wells and incubated for 1 hour at room temperature, then a washing step is performed to remove unbound material. Substrate is added and incubated for 10 minutes at room temperature. In the presence of bound H . pylori antigens, a color develops. Finally, a stop solution is added, and the results are read spectrophotometrically (450 nm). According to the manufacturer's instruction, the cutoff values are as follows: less than 0.140, negative (i.e., no H. pylori antigens in the stool samples, patients not infected); 0.140 to 0.159, equivocal-indeterminate (according to the manufacturer's instruction, indeterminate results should be repeated; if the repeated result is equivocal, a new specimen should be obtained); and greater than or equal to 0.160, positive (i.e., H. pylori antigens are present in the stool samples, patients infected). Such a test, which detects bacterial antigen in an ongoing infection, theoretically is useful not only for screening, but also as an early predictor of successful treatment. The authors consider briefly the currently available evidence supporting a possible role for this noninvasive diagnostic test. There are relatively few studies evaluating HpSA, most of which evaluate limited populations, assessing diagnostic accuracy in terms of sensitivity and specificity compared with more standardized techniques, and this is even more the case for assessing patients after eradication. An exception is a European multicenter study carried out in 11 endoscopic units with a specific interest in H. pylori infection throughout E u r ~ p e . 'The ~ first part of this prospective study was planned to assess the accuracy of HpSA compared with standardized technique; the authors enrolled 501 consecutive patients (not previously treated for H. pylori infection), who underwent endoscopy with multiple biopsies (for histology in antrum and corpus, rapid urease test, and culture) and I3C-urea breath test. A stool sample was collected from all patients. According to guidelines for clinical trialP in H. pylori infection, patients were considered H. pylori positive if at least two tests were positive; if culture alone was positive, in view of its absolute specificity, the patient also was considered positive. Using this gold standard, the overall sensitivity and specificity of the two noninvasive tests were 94% (confidence
920
VAIRAetal
Table 1. PROSPECTIVE EUROPEAN MULTICENTER STUDY SENSITIVITY AND SPECIFICITY OF HELlCOBACTER PYLORI STOOL ANTIGEN COMPARED WITH 13C-UREA BREATH TEST BEFORE AND AFTER TREATMENT ~~
~~~
Before Treatment (n = 501)
Sensitivity (“h) Specificity (%) HpSA
=
After Treatment (n= 133)
HpSA
“C-UBT
HpSA
‘3C-UBT
94.3 91.8
95.3 97.7
92.3 96.2
88.5 99.4
H.pylori stool antigen; W-UBT
=
’jC-urea breath test.
interval [CI], 90.6 to 96.6) and 91.8% (CI, 87.3 to 95.1) versus 95.3% (CI, 92.2 to 97.5) and 97.7% (CI, 94.8 to 99.3) for HpSA and 13C-ureabreath test (Table 1).In this study, a clear-cut result was obtained for most patients, with only 10 of 501 (2%) equivocal results. Of the 501 patients enrolled, 279 were found to be H. pylori positive, were given an eradicating treatment, and were asked to return 4 weeks after therapy for follow-up endoscopy, 13C-urea breath test, and HpSA. The authors have reported on 206 patients followed up: 133 by endoscopy, 13C-urea breath test, and HpSA and 73 by 13C-ureabreath test and HpSA only.2oIn the 133 patients (a population numerically important for a follow-up study) who underwent endoscopy, the overall sensitivity and specificity of HpSA and 13C-urea breath test were 92.3% and 96.2% versus 88.5% and 99.1% (see Table 1). The test gave clear-cut results in most patients: 132 of 133 (99.2%). Most of the remaining clinical studies on this test so far have been only as abstracts? In most cases, these studies confirm the excellent performance of the HpSA before and after therapy (although most studies involved limited populations); a few reports obtained considerably lower performance in terms of sensitivity and specificity, highlighting the need for further large validation studies (Table 2). In addition to evaluating the accuracy of the HpSA test compared with other techniques, other issues are being addressed by investigators throughout the world, such as the possible influence of concomitant use of proton-pump inhibitors (PPIs),” the kinetics of stool antigen during and after therapy,13,25 the possibility of cross-reaction with antigen from bacteria different from H. pylori,16 the possible use in pediatric populations,” the possibility that HpSA may detect rapid urease test-negative and histology-negative H. pylori-infected patients? and the cost-effectiveness of the test.= Vakil et alZ4evaluated the possible effect of concomitant use of PPIs on stool antigen secretion and found no differences between the value observed in 10 patients receiving PPIs (0.55k0.32) and 45 patients not receiving PPIs (0.61720.135). van’t Hoff et alZ5used the HpSA test to monitor the disappearance of H. pylori infection in the stomach during and shortly after eradication in a subgroup of 19 patients enrolled in the previously mentioned European multicenter study. The authors showed a fast decline of H. pylori antigen levels in the first 3 days after the start of the therapy, with all but two patients being below the cutoff value at 5 days. These results led the authors to conclude that if these data are confirmed by further studies, detection of HpSA would change dramatically the management of H. pylori patients. The same topic was addressed by Petersen et all3 who investigated 20 H. pyloripositive patients who were asked to provide a stool sample before initiating “References 1-3, 5, 8, 10, 12-14, 16, 24-26.
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ACCURATE DIAGNOSIS OF HELICOBACTER PYLORI
Table 2. SENSITIVITY AND SPECIFICITY OF HELlCOBACTER PYLORl STOOL ANTIGEN’ Before Treatment (n) Author
Braden et all Costa et a12 Feliciangeli et a13 Monteiro et all2 vakii24 Wirtheim et alZ6 Grubel et a15 Kim et a18 Lujan et all0 Sander et all4
After Treatment (n)
Sensitivity
Specificity
Sensitivity
Specificity
(W
@Y
(W
(W
95 (54) 94 (124) 80 (91) 87 (97) 90 (41) 96 (73) 100 (19) 100 (41)
-
-
90 (45)
80 (45)
-
-
93 97 58.8 88.4 87 83 88 87.4 97.3 98
(54) (124) (91) (97) (41) (73) (19) (41) (38) (137)
-
95 (137)
-
-
100 (14)
100 (14)
-
-
-
95 (26)
100 (26)
-
-
*Abstracts presented at the Digestive Disease Week (DDW), 1999 (see individual references).
eradicating therapy, then every other day for 2 weeks. In the 17 patients who completed the study, HpSA levels in stool usually increased, with a peak seen between 2 and 5 days, after which it decreased to undetectable levels if the treatment was successful. This peak in HpSA levels in the first 2 to 5 days of therapy was considered to be owing to the bactericidal effect of the antibioticreleasing antigen from the killed bacteria. The authors found that if the treatment was unsuccessful, the HpSA increased to approximately the same levels as seen initially over a few days. Taylor et all6evaluated the HpSA for possible cross-reactivateswith Helicobacter species other than H. pylori (including H . canis, H . rodentium, H . felis, H . pullorurn, H. cineady, H. fenneliae, H. bilis, H. hepaticus, H. rappini, and H . bizzozzeroni). The study was carried out in animal models using feces from Helicobacterinfected mice, ferrets, and cats. The authors found that other Helicobacter species, some of which have been reported in human disease, react in the H . pylori antigen capture assay and concluded that these data require confirmation in human studies to determinate the relevance of these findings in a clinical setting. Scant data are available on pediatric populations; however, at least two studies6, have included children. Both of these studies found a good performance of the stool test, and Kim et a18 concluded that the technique may be a substitute for endoscopy, especially in children and some patients unable to undergo endoscopic examination. Hsu et a16investigated four H. pylori-positive patients undergoing eradication treatment. In these patients, stool samples were collected every other day during antibiotic treatment and at 4 weeks after treatment, and the eradication was confirmed by 13C-urea breath test, biopsy specimens from antrum and corpus for culture, CLO test, and histology. Hsu et a16 found that at 4 weeks incomplete eradication in two patients resulted in positive urea breath test and HpSA but negative endoscopy tests, and they concluded that larger scale studies are needed to verify the cost-effectiveness of HpSA as a noninvasive test for eradication. The topic of cost-effectiveness of HpSA was addressed by Vakil,= who modeled a study on the cost-effectiveness of stool testing, urea breath test, office serology, enzyme-linked immunosorbent assay serology, and whole-blood testing in the initial diagnosis of the H . pylori infection and the cost-effectiveness
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VAIRAetal
of urea breath test and stool studies in patients given eradication therapy. In this mathematical model, VakiP found that before therapy the stool test compared favorably with the other technique assuming a prevalence of H. pylori infection of 30% in dyspeptic patients, and when the prevalence of infection dropped to 19% the stool test was the most cost-effective because of its higher specificity than office-based blood or serum tests. In posttherapy testing, the stool test was cost-effective compared with 14C-ureaand 13C-ureabreath test with an average cost of $183 2 197 versus 213 5 9 9 versus 369 5 105. The conclusions were that (1) stool tests are cost-effective alternatives to office-based serology testing, and (2) their most promising role is in posttreatment assessment. SUMMARY
From the limited data available, it seems that the H. pylori stool assay represents a highly accurate diagnostic tool to detect H. pylori infection before and shortly after therapy. As a test that is noninvasive, accurate, simple, and cost-effective, the H. pylori stool assay has the potential to become the preferred diagnostic tool in many different clinical settings from epidemiologic studies to pediatric investigations, from pre-endoscopic screening policies to posttherapy monitoring. References 1. Braden B, Dietrich CF, Teuber G, et al: New antigen test in stool detects Helicobacter pylori infection: Non invasive diagnostic tool for therapy control. Gastroenterology 116A127, 1999 2. Costa F, Bellini M, Mummolo G, et al: A new enzyme immunoassay to detect Helicobacter pylori (HI') antigens on stool specimens: Diagnostic accuracy before and after eradicating treatment. Gastroenterology 116:A139, 1999 3. Feliciangeli G, Macarri G, Di Sario A, et al: Diagnostic accuracy of PCR in the detection of Helicobacter pylori infection. Gastroenterology 116:A160, 1999 4. Gasbarrini A, Franceschi F, Gasbarrini G, et al: Extraintestinal pathology associated with Helicobacter infection. Eur J Gastroenterol Hepatol9:231-233, 1997 5. Grubel P, Franck RW, Willis DH, et ak HpSA enzyme immunoassay for the detection of H pylori from stool samples collected from a field in Egypt. Gastroenterology 116A177, 1999 6. Hsu RK, Solnick J, Ruebner B: Premier HpSA Helicobacter pylori stool antigen may detect CLO and histology negative infection after antibiotic therapy: A prospective study. Gastroenterology 116A191, 1999 7. International Agency for Research on Cancer, World Health Organisation: Infection with Helicobacter pylori. In: Schistosomes, Liver Flukes and Helicobacter pylori. Lyon, IARC, 1994, pp 177-202 8. Kim PS, Lee J, Pai SH, et al: Detection of Helicobacter pylori antigen in stoolmby ELISA. Gastroenterology 116:A215, 1999 9. Logan RPH: The urea breath test. In Lee A, Mkgraud F (eds): Helicobacter pylori: Technique for Clinical Diagnosis and Basic Research. London, WB Saunders, 1996, PP 74-81 10. Lujan M, I'amos S, Llucian R, et al: Sensitivity and specificity of H pylori antigen in faeces. Gastroenterology 116:A245, 1999 11. Megraud F How should Helicobacter pylori infection be diagnosed? Gastroenterology 112:S9>S98, 1997 12. Monteiro L, De Mascarel A, Barberis C, et al: What is the performance of the Premier Platinum HpSA test in comparison to other invasive tests for the detection of Helicobacter pylori infection? Gastroenterology 116A258, 1999
ACCURATE DIAGNOSIS OF HELICOBACTER PYLORI
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13. Petersen JR, Adesoji 8, Okorodudu A, et al: Using the Helicobacter pylori stool antigen (HpSA) in monitoring antibiotic treatment response of Helicobacter pylori. Gastroenterology 116A281, 1999 14. Sander JO, van Zanten V, Bleau BL, et al: Helicobacter pylori stool antigen test (HpSA): Use for detection of infection and eradication. Gastroenterology 116:A3639, 1999 15. Technical annex: Tests used to assess Helicobacter pylori infection: Guidelines for clinical trials in Helicobacter infection. Working party of the European Helicobacter pylori Study Group. Gut 4l(suppl2):Sl(rS18, 1997 16. Taylor NS, Esteves M, Fox JG: Cross reactivity with Helicobacter species assayed with a Helicobacter pylori antigen capture assay. Gastroenterology 116:A830, 1999 17. Thomas JE, Gibson GR, Darboe MK, et al: Isolation of Helicobacter pylori from human faeces. Lancet 340:1194-1195, 1992 18. Vaira D, et al: Prospective screening of dyspeptic patients by Helicobacter pylori serology: A safe policy? Endoscopy 29:595-601, 1997 19. Vaira D, Malfertheiner P, MCgraud F, et al, and European Helicobacter pylori HPSA Study Group: Diagnosis of Helicobacter pylori infection using a novel, noninvasive antigen based assay in a European multicentre study. Lancet 354:30-33, 1999 20. Vaira D, Malfertheiner P, MCgraud P, et al, and European HpSA Study Group: Non invasive tests for monitoring Helicobacter pylori (HP). European Multicentre Study. Gastroenterology 116:A341, 1999 21. Vaira D, Miglioli M, Mule P, et al: Prevalence of peptic ulcer in Helicobacter pylori positive blood donors. Gut 35:309-312, 1994 22. Vaira D, Stanghellini V, Menegatti M, et al, and The Italian Helicobacter Study Group: IgG ELISA antibodies and detection of Helicobacter pylori in elderly patients. Lancet 1~269-270,1996 23. Vakil N: Cost effectiveness of non invasive testing methods for H pylori before in dyspeptic patients. Gastroenterology 116:AlOO, 1999 24. Vakil N, Affi A, Sundaram M, et al: Prospective blinded evaluation of the accuracy of a stool test for the detection of H pylori. Gastroenterology 116:A342, 1999 25. van’t Hoff BMW, Vaira D, van der Ende A, et al: A novel non invasuve test to assess Helicobacter pylori (Hp) shortly after eradicating treatment. Gastroenterology 116:A341, 1999 26. Wirtheim E, Yahav J, Mauz E, et al: The sensitivity and specificity of a novel enzyme immunoassay (Meridian HpSA) for the detection of Helicobacter pylori antigen stool specimens. Gastroenterology 116:A355, 1999
Address reprint requests to Din0 Vaira, MD Clinica Medica I Universiti di Bologna Policlinico S. Orsola V. Massarenti g 40138 Bologna Italy e-mail: [email protected]