University of Groningen The significance of Helicobacter ... · The significance of Helicobacter pylori in the approach of dyspepsia in primary care Contents Chapter 1 9 Introduction

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The significance of Helicobacter pylori in the approach of dyspepsia in primary careArents, Nicolaas Lodevikus Augustinus

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The significance of Helicobacter pylori in theapproach of dyspepsia in primary care

N.L.A. Arents

2003

De SENSE studie (Strategy: ENdoscopy versus SErology) is mogelijk gemaakt

door : Aventis Pharma B.V.

Het drukken van dit proefschrift is financieel ondersteund door :

Mediphos Medical Supplies B.V.

Oxoid B.V.

GlaxoSmithKline B.V.

ABBOTT B.V.

bioMérieux B.V.

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Cover illustration : painting by Suzanne C.B. Prins

Printed by Stichting Drukkerij C. Regenboog, Groningen, The Netherlands

RIJKSUNIVERSITEIT GRONINGEN

The significance of Helicobacter pylori in theapproach of dyspepsia in primary care

Proefschrift

ter verkrijging van het doctoraat in deMedische Wetenschappen

aan de Rijksuniversiteit Groningenop gezag van de

Rector Magnificus, dr. F. Zwarts,in het openbaar te verdedigen op

woensdag 18 juni 2003om 14.15 uur

door

Nicolaas Lodevikus Augustinus Arentsgeboren op 20 augustus 1971

te Rheden

Promotores prof. dr. J.H. Kle ibeukerprof. dr. J.E. Degener

Co-promotores dr. J.C. Thijsdr. A.A. van Zwet

Beoordelings commissie prof. dr. J.B.M.J. Jansenprof. dr. E.J. Kuipersprof. dr. H.A. Verbrugh

ISBN-nummer : 90-367-1788-4

Contents

The significance of Helicobacter pylori in the approach of

dyspepsia in primary care

Contents

Chapter 1 9

Introduction and outline of the thesis.

Chapter 2 15

A rational approach to uninvestigated dyspepsia in primary care. Review of the literature.

Postgraduate Medical Journal 2002; 78: 707-716.

Chapter 3 51

The approach of dyspepsia in primary care. A randomised trial comparing “test-and-treat”

with prompt endoscopy.

In press, Archives of Internal Medicine.

Chapter 4 73

The accuracy of the Helicobacter pylori Stool Antigen test (HpSA test) in diagnosing

Helicobacter pylori in treated and untreated patients.

European Journal of Gastroenterology & Hepatology 2001;13:383-386.

Chapter 5 83

Does the declining prevalence of Helicobacter pylori unmask patients with idiopathic

peptic ulcer disesase ?

Submitted for publication.

Chapter 6 93

Changes in the prevalence of metronidazole and clarithromycin resistance and its influence

on the efficacy of triple therapies in Helicobacter pylori.

In part accepted for publication in Journal of Antimicrobial Chemotherapy.

Chapter 7 105

The importance of vacA, cagA, and iceA genotypes of Helicobacter pylori infection in

peptic ulcer disease and gastroesophageal reflux disease.

American Journal of Gastroenterology 2001; 96:2603-2608.

Chapter 8 119

The value of PCR and serology based methods in determining the presence of the cagA

gene in Helicobacter pylori infection.


Chapter 9 131

Implications of the simultaneous presence of metronidazole-susceptible and -resistant

Helicobacter pylori colonies within a single biopsy specimen.

European Journal of Clinical Microbiology & Infectious Diseases 2001; 20:418-420.

Chapter 10 139

Summary and general conclusions.

Chapter 11 145

Samenvatting en algemene conclusies.

Dankwoord 153

9

Chapter 1

Introduction and outline of the thesis.

Chapter 1

10

Introduction and outline of the thesis

Dyspepsia is a complex of symptoms believed to originate from the upper gastrointestinal tract

[1-4]. These symptoms are very common in Western societies and about 30% of the general

population experiences at least one period of more or less severe dyspepsia during any given year

[5-7]. Despite this high prevalence of dyspepsia, only a minority of the patients will eventually

seek medical attention [5,6]. Nevertheless, dyspepsia accounts for 1 - 4% of all consultations in

primary care [8-10]. Most of these patients are not referred for further investigation and are

managed solely by their general practitioner [8,11,12]. Although general practitioners are

frequently consulted for dyspepsia, the optimal approach towards this problem has not been

established sofar. Defining the optimal approach for dyspepsia is difficult since dyspepsia is not a

diagnosis but may result from various underlying conditions ranging from potentially life

threatening disorders (gastric cancer, peptic ulcer disease (PUD)) to functional diseases with a

benign course. Unfortunately, apart from alarm symptoms, other symptoms relate poorly to

underlying disease [6] and are of limited value for the choice of therapy [13,14]. Several strategies

have been proposed to manage patients with dyspepsia without alarm features in primary care. The

discovery of Helicobacter pylori (H. pylori) and the acknowledgement of its important etiological

role in several diseases of the upper alimentary tract have added even more possibilities to

approach dyspepsia. The main strategies currently considered are: 1. Symptom guided empirical

treatment, 2. Direct referral for endoscopy, 3. Non-invasive testing for H. pylori and subjecting the

H. pylori-positive patients to endoscopy (“test-and-scope”), and 4. Non-invasive testing for

H. pylori and treatment of the infection in H. pylori-positive patients (“test-and-treat”). Both

strategies based on screening for H. pylori infection have been studied in secondary care settings,

but studies evaluating its efficacy in a primary care setting are lacking. Chapter two considers the

rationale for the various strategies in primary care, based on the available data in literature. In

chapter three the results of a randomised clinical trial are reported comparing the “test-and-treat”

strategy to prompt endoscopy. It was hypothesised that only a minority of the patients approached

by the “test-and-treat” strategy would be referred for endoscopy and that significant disease would

not be missed. Moreover, this study was designed to enable a comparison of symptom resolution,

quality of life, patient satisfaction and medical cost per patient in both study groups.

In order to successfully implement a strategy that is based on screening for H. pylori, some

conditions have to be met. First of all, the non-invasive test used to detect the presence of H. pylori


11

has to be accurate. Chapter four reports the results of a study evaluating a recently introduced non-

invasive method to detect the presence of H. pylori infection by testing for H. pylori antigens in

faecal samples. Secondly, the prevalence of H. pylori has to be sufficiently high in the dyspeptic

population, but especially in patients with PUD, in whom treatment of the infection is most

beneficial. It has recently been suggested that an increasing number of patients with PUD are not

infected with H. pylori and that the decreasing prevalence of H. pylori unmasks this so-called

idiopathic PUD (not associated with H. pylori, NSAID use, or any other identifiable cause) [15].

In Chapter five a study is reported in which we examined the prevalence of idiopathic PUD and

trends over a period of 8 years. Thirdly, since it is well known that antibiotic resistance in

H. pylori (mainly to metronidazole and clarithromycin) has a significant impact on the efficacy of

the frequently used triple regimens [16,17], knowledge about the prevalence of resistance to

metronidazole and clarithromycin is important before prescribing these drugs. Chapter six

describes the prevalence of metronidazole and clarithromycin resistance over an eight year period

in the province of Drenthe. Moreover, the calculated effect of different prevalences of resistance

on the efficacy of two commonly used triple regimens is described .

The second part of the thesis focuses on several microbiological aspects of the highly diverse

H. pylori species. Most H. pylori infected patients carry a unique strain [18,19] and even within a

single patient different H. pylori strains can be encountered [20,21]. It has been suggested that

genetic differences among H. pylori strains are related to the different sequelae of the infection.

Chapter seven presents the results of a study into the relation between several virulence factors of

H. pylori and clinical outcome. Chapter eight evaluates a new rapid test to detect the presence of

CagA antibodies to the virulence factor cagA as compared to polymerase chain reaction (PCR) on

biopsy specimens and the OroVax CagA protein recombinant based ELISA. Finally in chapter

nine we studied patients carrying both metronidazole susceptible and resistant H. pylori bacteria.

The question was whether this phenomenon was based on a multiple strain infection or not.

Chapter ten summarises this thesis and presents the general conclusions that were drawn from all

studies.

Chapter 1

12

References

1. Crean GP, Card WI, Beatti AD, J. HR, James WB, Knill-Jones RP et al. Ulcer-like dyspepsia.

Scandinavian Journal of Gastroenterology 1982; 17 (suppl 79): 9-15.

2. Colin-Jones DG. Management of dyspepsia: report of a working party. Lancet 1988; 1: 576-579.

3. Barbara L, Camilleri M, Corinaldesi R, Crean GP, Heading RC, Johnson AG et al. Definition

and investigation of dyspepsia. Digestive Diseases and Science 1989; 34: 1271-1276.

4. Heading RC . Definitions of Dyspepsia. Scandinavian Journal of Gastroenterology 1991; 26 (suppl

182): 1-6.

5. Jones RH, Lydread SE, Hobbs FDR, Kenkre JE, Williams EI, Jones SJ et al. Dyspepsia in

England and Scotland. Gut 1990; 31: 401-405.

6. Penston JG, Pounder RE. A survey of dyspepsia in Great Britain. Alimentary Pharmacology &

Therapeutics 1996; 10: 83-89.

7. Bernersen B, Johnsen R, Straume B, Burhol PG, Jenssen TG, Stakkevold PA. Towards a true

prevalence of peptic ulcer: the Sorreisa gastrointestinal disorder study. Gut 1990; 31: 989-992.

8. Jones R, Lydeard S. Prevalence of symptoms of dyspepsia in the community. British Medical

Journal 1989; 298: 30-32.

9. Heikkinen M, Pikkarainen P, Takala J, Julkunen R. General practicioners' approach to dyspepsia.

Scandinavian Journal of Gastroenterology 1996; 31: 648-653.

10. Knill-Jones RP. Geographical differences in the prevalence of dyspepsia. Scandinavian Journal of

Gastroenterology 1991; 26 (suppl 182): 17-24.

11. Muris JWM, Starmans R, Fijten GH, Crebolder HFJM, Krebber TFWA, Knottnerus JA.

Abdominal pain in general practice. Family Practice 1993; 10: 387-390.

12. Warndorff DK, Knottnerus JA, Huijnen LGJ, Starmans R. How well do general practitioners

manage dyspepsia ? Journal of the Royal College of General Practitioners 1989; 39: 499-502.

13. Adang RP, Ambergen AW, Talmon JL, Hasman A, Vismans JF, Stockbrugger RW. The

discriminative value of patient characteristics and dyspeptic symptoms for upper gastrointestinal

endoscopic findings: a study on the clinical presentation of 1,147 patients. Digestion 1996; 57: 118-

134.

14. Bytzer P, Schaffalitzky de Muckadell OB. Prediction of major pathologic conditions in dyspeptic

patients referred for endoscopy. A prospective validation study of a scoring system. Scandinavian

Journal of Gastroenterology 1992; 27: 987-992.

15. Higuchi K, Arakawa T, Fuijwara Y, Uchida T, Tominaga K, Watanabe T et al. Is Helicobacter

pylori-negative duodenal ulcer masked by the high prevalence of H.pylori infection in the general

population ? American Journal of Gastroenterology 1999; 94: 3083-3084.


13

16. Houben MH, Van Der BD, Hensen EF, Craen AJ, Rauws EA, Tytgat GN . A systematic review of

Helicobacter pylori eradication therapy. The impact of antimicrobial resistance on eradication rates.

Alimentary Pharmacology & Therapeutics 1999; 13: 1047-1055.

17. van der Wouden EJ, Thijs JC, van Zwet AA, Sluiter WJ, Kleibeuker JH. The influence of in

vitro nitroimidazole resistance on the efficacy of nitromidazole containing anti-Helicobacter pylori

regimens: a meta-analysis. American Journal of Gastroenterology 1999; 94: 1751-1759.

18. Owen RJ, Bickley J, Costas M, Morgan DR. Genomic variation in Helicobacter pylori: application

to identification of strains. Scandinavian Journal of Gastroenterology 1991; 181: 43-50.

19. Akopyanz N, Bukanov NO, Westblom TU, Kresovich S, Berg DE. DNA diversity among clinical

isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Research

1992; 20: 5137-5142.

20. Jorgensen M, Daskalopoulos G, Warburton V, Mitchell HM, Hazell SL. Multiple strain

colonization and metronidazole resistance in Helicobacter pylori-infected patients: identification

from sequential and multiple biopsy specimens. Journal of Infectious Diseases 1996; 174: 631-635.

21. Prewett EJ, Bickley J, Owen RJ, Pounder RE. DNA patterns of Helicobacter pylori isolated from

gastric antrum, body and duodenum. Gastroenterology 1992; 102: 829-833.

15

Chapter 2

A rational approach to uninvestigated dyspepsia in primary care.

Review of the literature.

Nicolaas L. A. Arents 1, Jacob C. Thijs 2, and Jan H. Kleibeuker 3

1 Regional Public Health Laboratory, Groningen / Drenthe2 Department of Internal Medicine, Bethesda Hospital, Hoogeveen3 Department of Gastroenterology, University Hospital, Groningen

Postgraduate Medical Journal 2002; 78: 707-716.

Chapter 2

16

Abstract

In this paper the rationale and limitations are analysed of the four most important approach

strategies to dyspepsia in primary care (empiric treatment, prompt endoscopy, “test-and-scope”,

and “test-and-treat”). It is concluded that in the absence of alarm symptoms, a “test-and-treat”

approach is currently the most rational approach provided that three conditions are met: 1. a highly

accurate test should be used, 2. the prevalence of H. pylori in the population should not be too low

and 3. an effective anti-H. pylori regimen should be prescribed taking sufficient time to instruct

and motivate the patient.

Introduction

Dyspepsia is not a diagnosis, but merely a cluster of symptoms believed to be referable to the

upper gastrointestinal tract [1-4]. It is very common in the adult western population with

prevalence rates ranging from 19 up to 41% in several epidemiological studies [5-17]. Although

most dyspeptics do not seek medical attention, half of them regularly use over the counter drugs

[10,12,16]. About one out of every four subjects with dyspepsia consults his general practitioner

[9,10,13,16,18] and these visits account for 1- 4% of all consultations in primary care [9,12,19].

Twenty five percent of these patients are referred for further investigations (i.e. endoscopy,

ultrasonography, etc) or to a secondary care physician (about 10%) but the majority of the patients

are managed empirically by their general practitioner [9,10,20-22]. Altogether, dyspepsia is an

important health issue and constitutes a significant clinical problem in primary care.

The objective of general practitioners in the approach of dyspeptic patients is to abolish or

reduce symptoms, either by the detection and treatment of underlying disease or by symptomatic

treatment or mere reassurance of the patient. Secondary objectives are the timely diagnosis of

significant disease, avoidance of over-treatment, and containment of costs (table 1). The optimal

approach to achieve these goals, however, is still a matter of discussion, even though several

guidelines have been formulated [23-25]. This current lack of agreement is related to several

points, starting with the definition of dyspepsia. According to the recent Rome consensus [26] the

term dyspepsia refers to persistent or recurrent upper abdominal pain or discomfort, supposed to be

referable to the upper gastrointestinal tract. The consensus meeting excluded patients with

heartburn or acid regurgitation as the predominant symptom, as these symptoms were thought to

A rational approach to uninvestigated dyspepsia in primary care

17

Table 1.

Objectives in the approach of the dyspeptic patient in primary care

• Abolishment or reduction of symptoms

• Timely diagnosis of significant disease

• Avoidance of over-treatment

• Containment of medical and non-medical costs

be predictive of gastro-esophageal reflux disease (GERD) [27,28]. Nevertheless, many studies

ondyspepsia used other definitions and some do include patients with predominant heartburn [9-

11,13,16]. Secondly, the optimal approach of the dyspeptic patient depends on the prevalences of

the underlying causes of dyspepsia. These prevalences differ between various parts of the world.

For example, in South East Asia, where gastric cancer is a common condition [29], the optimal

approach is likely to be different from that in the Western world [30]. Thirdly, the role of some

conditions in causing dyspeptic symptoms is still debated. This is especially true for the

relationship between Helicobacter pylori (H. pylori) gastritis and dyspepsia without an

endoscopically identifiable cause (functional dyspepsia; FD). Finally, what is the most cost-

effective approach largely depends on the local availability and costs of medical provisions

(especially endoscopy).

The primary care physician can choose between the following strategies for the approach of

uninvestigated dyspepsia: 1. Symptom guided empirical treatment, 2. Direct referral for

endoscopy, 3. Non-invasive testing for H. pylori and subjecting the H. pylori-positive patients to

endoscopy (“test-and-scope”), and 4. Non-invasive testing for H. pylori and treatment of the

infection in H. pylori-positive patients (“test-and-treat”). The aim of this review is to evaluate the

rationale behind each strategy and to weigh the available clinical evidence. It focuses on dyspepsia

as encountered in primary care and the situation in the Western world. It does not discuss the

approach of the patients with mild symptoms of short duration, which usually can be managed by

reassurance and simple lifestyle advice. Nor does it consider the approach of patients with

heartburn or acid-regurgitation as the only or predominant symptom. It is recognised, however,

that heartburn is frequently part of the dyspeptic syndrome [26]. Finally, patients with alarm

symptoms (table 2) and patients using non-steroidal anti-inflammatory drugs (NSAID’s) are not

subject of this review. Patients with alarm symptoms should be subjected to endoscopy without

Chapter 2

18

delay. In patients using NSAID’s, this drug should be discontinued if at all possible before

considering further steps.

Table 2.

Sinister symptoms in the dyspeptic patient, necessitating endoscopy

• Unintentional weight loss

• Iron deficiency anaemia

• Gastro-intestinal bleeding

• Dysphagia

• Odynophagia

• Previous gastric surgery

• Persistent vomiting

• Epigastric mass

Differential diagnosis in uninvestigated dyspepsia

Dyspepsia can be ascribed to several conditions. Only a few studies examined their relative

prevalences in a primary care population. In three studies all patients consulting their general

practitioner for dyspeptic symptoms were referred for endoscopy. Reflux oesophagitis was found

in 6-24%, peptic ulcer disease (PUD) in 13-16%, malignant disease in 1-2%, and in more than

60% of the patients no abnormalities were found at endoscopy [31-33] the latter patients being

usually diagnosed as having FD. More data are available from studies evaluating “open access

endoscopy”. In “open access endoscopy” the general practitioner directly refers the patients for

upper gastrointestinal endoscopy without prior evaluation by a specialist. Data from these studies

should, however, be considered with caution. The patients represent a selected population, as no

more than 25% of the patients in primary care are referred for further investigations [9,10]. In

these studies oesophagitis was diagnosed in 2-31%, PUD in 13-28%, a malignancy in 0.2-4.0%

and endoscopy revealed no visible disease in 35-89%. In table 3 the data from the three primary

care studies and the “open access endoscopy” studies with over 400 participating patients each are

summarised [31-50]. Considering these data, it is noteworthy that the ranges of frequencies of the

various endoscopic diagnoses in the studies evaluating “open access endoscopy” are very much

alike those in the primary care studies. This suggests that clinical judgement does not adequately


19

Table 3.

Prevalences of endoscopically encountered diseases in dyspeptic patientsn reflux

oesophagitis(%)

peptic ulcerdisease

(%)

malignancy

(%)

functionaldyspepsia

(%)unselected primary care patientsHeikinnen et al. (31) † 400 15 13 2.3 70Kagevi et al. (32) † 172 6 13 1.2 79Hansen et al. (33) 612 24 16 0.7 59

range 6-24 13-16 0.7-2.3 59-79weighted mean 18 15 1.3 66

open-acces endoscopy studies with > 400 patientsAyoola et al. (34)† 10112 11 17 1.5 70Hungin et al. (35) 6744 17 15 0.6 67Hallisey et al. (36) 2659 19 16 2.7 62Fedail et al. (37) 2500 8 18 2.2 71Mansi et al. (38) 2086 5 13 1.5 81Holdstock et al. (39) 1805 9 14 2.0 75Fjosne et al. (40) † 1275 10 23 4.0 62Capurso et al. (41) 1153 3 8 0.6 89Stanghellini et al. (42) 1057 9 21 0.6 69Davenport et al. (43) 1041 na 24 2.6 naNyren et al. (44) 972 3 23 0.3 74Gear et al. (45) 968 5 17 2.3 76Johannessen et al. (46)†

930 15 17 1.0 67

Bytzer et al. (47) 878 2 15 1.4 82Talley et al. (48) 820 14 23 3.4 60Williams et al. (49) 686 14 28 2.5 56Saunders et al. (50) † 559 31 23 0.2 46

range (%) 2 - 31 13 - 28 0.2 - 4.0 35 - 89weighted mean (%) 12 17 1.6 70na = data not available

† = in these studies more than one diagnosis was allowed in a single patient. Therefore, as

functional dyspepsia was defined as the absence of either reflux oesophagitis, peptic ulcer disease

or malignancy, this may have led to an underestimation of the percentage of patients with

functional dyspepsia (within a total of 100%).

Chapter 2

20

select patients at higher risk for underlying pathology which is in line with other studies showing

that it is impossible to predict endoscopic lesions in dyspeptic patients only from the clinical

presentation [16,47,51,52]. It is recognized, however, that in daily practice clinical judgement is

not the only consideration to refer a patient for endoscopy, and other reasons, like patients fear for

cancer, may be involved. It is also noteworthy that the prevalences of endoscopic diagnoses vary

considerably among the studies. This is probably explained by differences in inclusion criteria and

by geographic differences, but prevalences are also changing. Several studies have shown that

during the last decades the prevalences of GERD and adenocarcinoma of the oesophagus and

cardia have been increasing rapidly in the western world, together with decreasing prevalences of

PUD and distal gastric cancer [53-55]. The decreasing prevalence of H. pylori infection is

generally considered to contribute to these epidemiological changes [56]. Considering these

varying and changing prevalences of underlying conditions in dyspepsia, it is obvious that the

optimal approach of the dyspeptic patient may be different between the various geographic regions

and may also change with time.

The role of H. pylori in dyspepsia

In the years following the first description of H. pylori by Warren and Marshall in 1983 [57] it

has become clear that this gram-negative curved bacterium plays a major role in several conditions

of the upper gastrointestinal tract. There is ample evidence showing a causal relationship between

H. pylori infection and PUD. When patients using NSAID’s are excluded, the large majority of

PUD patients carry the infection [58] and eradication of H. pylori heals the disease [59-66] and

prevents its recurrence [61-64,66-69]. Moreover, H. pylori gastritis is the most important risk

factor for the subsequent development of PUD [70,71] a risk that has been estimated to be about

10% in a lifetime [72,73]. On the other hand, several recent studies show that not all PUD is

caused by H. pylori, even after exclusion of patients using NSAID’s [74-78]. The decreasing

prevalence of H. pylori in the western world is likely to cause a relative increase in the proportion

of these “idiopathic” peptic ulcers [76,79].

In addition to the causal relation with PUD, it has been shown that there is also a strong

association between H. pylori gastritis and the occurence of gastric cancer [80-89] and the

International Agency for Research on Cancer has classified H. pylori as a class I risk factor for

gastric cancer [90]. Moreover, gastric MALT lymphoma (mucosa associated lymphoid tissue


21

lymphoma) [91-93] and Ménétrier's disease of the stomach have been associated with H. pylori

[94-96]. These conditions, however, are rare, and therefore this association will not be a major

factor influencing decision making in the dyspeptic patient.

The relationship between H. pylori and FD has been debated for a long time and is still not

entirely clarified [97,98]. Several studies have suggested a higher prevalence of H. pylori in FD,

but others came to dissimilar conclusions [99,100]. Decisive evidence could be expected from

studies evaluating the effect of H. pylori eradication on symptoms in FD. Early studies were

flawed by methodological problems [101,102] but recently, four large well conducted randomised

placebo-controlled clinical trials with a follow-up period of 12 months have been published as full

paper [103-106]. The results, however, are contradictory (table 4). A recent meta-analysis included

these studies as well as five other studies [107-111] and suggested that there is a small but

significant benefit of about 10% of H. pylori eradication over placebo [112]. Another meta-

analysis, however, included other studies [109,113-117] apart from the ones listed in table 4 and

did not show any benefit [118].

H. pylori infection does not seem to be a causal factor in the pathogenesis of GERD [119-121].

Epidemiological data even suggest that the infection protects against this condition. While the

prevalence of H. pylori gastritis is declining in the western world, GERD and Barrett’s carcinoma

are becoming more common [53,122,123]. Moreover, some studies suggest that eradication of H.

pylori can induce GERD [124,125], although this has not been confirmed by others [126-128].

Immediate endoscopy or symptom guided empirical treatment

Endoscopy is the most appropriate investigation to detect pathological lesions in the upper

alimentary tract as well as the presence of H. pylori [129,130]. Immediate endoscopy in patients

with dyspepsia results in a definite diagnosis from the outset and ensures that the patient receives

the most appropriate treatment. It is evident that in most patients with dyspepsia no underlying

disease can be identified. Several studies, however, have shown that even then an endoscopy may

have its merits. A negative endoscopy may have a significant reassuring effect and may result in a

decreased use of medication and in fewer medical consultations [131-133]. Investigating all

dyspeptic patients by endoscopy, however, is not feasible in view of the high incidence of

dyspepsia and the limited availability of endoscopic facilities. Therefore, alternative approaches

have been suggested.

Chapter 2

22

Many dyspeptic patients consult their general practitioner mainly because of fear of possible

serious disease and sometimes mere reassurance may be sufficient [13,18,46,134]. If the

symptoms do not abolish spontaneously, it has been proposed to prescribe a trial of treatment,

reserving endoscopy for those patients who do not respond or whose symptoms recur after

stopping treatment. [20-22,135]. This strategy has been promoted by several organisations of both

primary and secondary care physicians [2,25,49,129,136]. The choice of the drug may be dictated

by the predominant symptom [2] but treatment most often consists of an acid suppressing drug

given during a fixed period. H2-receptor blocking agents (H2RA’s) are used predominantly,

although they are being gradually replaced by the more powerful proton pump inhibiting drugs

(PPI’s). Acid suppression is effective in GERD and PUD and a few studies have shown some

efficacy in FD [137-140]. Besides acid-suppressive drugs, prokinetics, especially cisapride, have

been advocated for the empirical treatment of uninvestigated dyspepsia. This drug is shown to be

of benefit in GERD [141-152] and FD [153-160], and may have some efficacy in PUD [161-164].

Recently, however, serious cardiac side effects of cisapride were reported and, for that reason, the

drug has been withdrawn from the US market [165].

Table 4.

Effect of H. pylori eradication on symptom improvement in patients with functional

dyspepsia: results of double blind placebo controlled studies with

1 year follow-up published as full-papers

n = anti-H. pylori treatment

symptom improvement (%)

placebo

symptom improvement

(%)

Blum et al. (106)

McColl et al. (105)

Talley et al.(104)

Talley et al. (103)

328

318

275

337

27.4

21

24.1

46

20.7

7*

21.8

50

*p = 0.03


23

Selection for endoscopy by non-invasive testing for H. pylori: the “test-and-scope” and “test-

and-treat” strategy

The incorporation of the H. pylori concept in the approach of uninvestigated dyspepsia has led

to the development of two major strategies: the “test-and-scope” strategy and the “test-and-treat”

strategy.

The “test-and-scope” strategy was first suggested by Sobala et al. in 1991 [166]. They

reasoned that dyspeptic patients under the age of 45 without alarm symptoms or NSAID use and

testing negative for H. pylori by non-invasive means have a very low probability for serious

underlying disease. Therefore, they postulated that these patients could safely be treated

empirically, without the need for an endoscopy. When this strategy was tested retrospectively in a

group of 293 patients referred for endoscopy, they found that the total number of endoscopies

performed would have been reduced by 25% in all age groups and by 48% in the age group below

45 years, without missing any peptic ulcer or malignancy. Other retrospective studies showed

comparable results with a reduction in the number of endoscopies between 11-35% when all

patients referred for endoscopy were considered, be it at the cost of missing up to 2% of the peptic

ulcers [167-175]. Patel et al. were the first to evaluate this strategy prospectively in 183

consecutive patients [176]. Seventy patients below the age of 45 without sinister symptoms or

NSAID use and testing H. pylori negative by serology were returned to their general practitioner

for symptomatic treatment without an endoscopy performed. Only three of these patients

eventually underwent endoscopy, resulting in an overall reduction in the number of endoscopies

by 37%. Moreover, endoscopy did not seem to influence subsequent symptom severity, quality of

life, and number of days lost from work when these non-endoscoped patients were compared with

an historic control group of H. pylori negative endoscoped subjects. Number of physician visits

and medication use were even less in these non-endoscoped patients. This is in accordance with

the study of Asante et al., who showed that endoscopy in sero-negative patients does not influence

quality of life, number of visits to the general practitioner or number of days lost from work [177].

Further reduction in the number of endoscopies could possibly be reached if H. pylori positive

dyspeptic patients would not be subjected to endoscopy, but would receive immediate anti-H.

pylori treatment : the “test-and-treat” strategy. In this way, patients with H. pylori related PUD

would be treated adequately without the need for an endoscopy. If the symptoms persist, the

patient can still be referred for endoscopy.

Chapter 2

24

Studies comparing the various strategies

In several studies the various strategies to approach the dyspeptic patient were compared by

computer-assisted decision analysis [178-185]. Decision analysis is a quantitative method for

estimating the outcome, both clinical and financial, of alternative management strategies. In such

an analysis calculations are based on data from the literature and on estimates by expert opinion.

Data in the model are often uncertain but recalculation of the outcome after varying a single

assumption over a reasonable range can be used to show to what extent different parameters

influence the conclusions. In that way these studies have identified factors, summarised in table 5,

that have a significant impact on the cost-effectiveness of any approach of dyspepsia. Most studies

concluded that a non-invasive approach, especially by a “test-and-treat” strategy, is likely to be the

most cost-effective [178,181-185], although this conclusion was heavily influenced by the costs of

endoscopy [179]. In these decision analytic studies, however, not all variables were incorporated

in the model and, some aspects with a possible impact on the outcome of a strategy were not

studied. Such aspects include a positive family history (PUD, cancer), the possible reassuring

effect of a negative endoscopy and the significance of detecting Barrett’s oesophagus, to name a

few. Therefore, such mathematical studies cannot replace prospective clinical trials.

Table 5.

Factors with significant impact on the cost-effectiveness of the various approaches in

patients with dyspepsia

• cost of endoscopy

• cost of therapy

• cost of physician visit

• prevalence of H. pylori in the dyspeptic population

• prevalence of PUD in H. pylori positive patients

• prevalence of underlying diseases in dyspepsia

• benefit of H. pylori eradication in patients with functional

• recurrence of dyspeptic symptoms in patients with (functional) dyspepsia after treatment


25

Just a few randomised trials have evaluated the various approaches of the dyspeptic patient in a

clinical setting [131,186-190]. Most of them compared a non-invasive approach, either the “test-

and-treat” strategy or empirical acid-suppressive treatment, with prompt endoscopy.

The “test-and-treat” strategy was evaluated in a limited number of studies [186-188,191].

Heaney et al. randomised H. pylori positive patients as determined by the 13C-Urea breath test

(13C-UBT) to either anti-H. pylori treatment or prompt endoscopy [186]. In the endoscopy group

only the PUD patients received anti-H. pylori treatment. At 1-year follow-up, only 27% of the

patients who were not endoscoped initially, had undergone endoscopy. The dyspepsia scores had

improved significantly more in the non-endoscoped patients than in those subjected to endoscopy.

Jones et al. compared the “test-and-treat” strategy with standard clinical care in primary care

[187]. In standard clinical care, only patients with proven PUD received anti-H. pylori treatment. It

was shown that during a follow-up of one year, only 12% of the patients in the “test-and-treat”

group eventually underwent endoscopy, as compared to patients receiving standard clinical care.

Clinical outcome was comparable in both groups, but medical costs in the “test-and-treat” group

were only about half of that in the control group. Lassen et al. compared the “test-and-treat”

strategy using the 13C-UBT with prompt endoscopy [188]. Again, in the endoscopy arm, only the

PUD patients received anti-H. pylori treatment. At the end, 40% of the patients in the “test-and-

treat” group had been referred for endoscopy. Symptom resolution, changes in quality of life, the

use of resources (medication, visits to outpatient clinics, days in hospital, visits to general

practitioner), and the number of sick-leave days were comparable in both study groups. Patients in

the endoscopy group, however, were more satisfied with the treatment and were more often

prescribed PPI’s. It was concluded that the “test-and-treat” strategy is as efficient and safe as

prompt endoscopy. Finally, Arents et al. compared the “test-and-treat” strategy using H. pylori

serology with prompt endoscopy [191]. In contrast to other studies, all H. pylori positive patients

received anti-H. pylori treatment in both study arms. At 1 year follow-up, 40% of the patients in

the “test-and-treat” group had been referred for endoscopy. Symptom resolution, improvement in

quality of life and patient satisfaction were comparable in both study groups.

The most important study comparing acid suppression to prompt endoscopy was performed by

Bytzer et al. [131]. In that study, patients were randomised to prompt endoscopy or to a trial of

empirical treatment with H2RA. Prompt endoscopy proved to be more cost effective and to cause

more patient satisfaction than empirical treatment with an H2RA. Laheij et al compared prompt

Chapter 2

26

endoscopy with an approach in which patients initially received empirical treatment with

omeprazole, followed by a “test-and-treat” strategy if symptoms persisted [189]. After 1-year

follow-up, 31% of the patients in the group receiving empirical treatment had been referred for

endoscopy. Clinical outcome was the same in both groups, but medical costs were lower in the

group at first subjected to empirical treatment. Finally, in one study, all four different strategies

were compared: initial treatment with acid suppression, “test-and-treat” , “test-and-scope”, and

immediate endoscopy [190]. In that study, prompt endoscopy was initially the most expensive

approach, but after a 1-year follow-up period it became the most cost-effective one as subsequent

costs were reduced.

Like the decision analytic studies, also these clinical studies have their limitations. They were

performed in a specific setting in which the factors, listed in table 5 all have their significant

influence on the outcome. These factors may have a diverse impact in different regions or clinical

settings. For instance, all studies were performed in a secondary care setting and the results may

not be applicable for the dyspeptic patient visiting his primary care physician. Moreover, all

studies used a specific treatment protocol. For example, in the two trials comparing a “test-and-

treat” strategy with prompt endoscopy, all patients testing positive for H. pylori in the “test-and-

treat” group received anti-H. pylori treatment whereas H. pylori positive patients in the endoscopy

group only received such treatment if they had endoscopic evidence of PUD denying FD patients

any possible benefit of anti-H. pylori treatment. Such a study design may have biased the results.

The rationale and limitations of the various approaches of the dyspeptic patient

The optimal approach of the dyspeptic patient in primary care is the one that best serves the

objectives listed in table 1. Frequently, none of the aforementioned strategies will positively serve

all these objectives and in clinical practice none of the strategies is always clearly superior to the

others. What is the most cost-effective strategy is determined by the factors enumerated in table 5

and the significance and impact of most of these factors may vary considerably between different

regions of the world. Even within a single region, however, it is not likely that a single strategy is

the best one in every patient. Sometimes a different approach should be chosen as dictated by the

characteristics of the individual patient (age, family history, comorbidity, fear of cancer etc.).

Therefore, it is the authors' view that, in order to be able to treat the dyspeptic patient in the most


27

cost-effective way, the primary care physician should be aware of the factors listed in table 5 and

understand the rationale and limitations of the various approaches of uninvestigated dyspepsia.

If one chooses to prescribe empirical treatment with an acid-suppressive agent, what can be

expected? It is evident that acid-suppressive therapy will be beneficial in patients with PUD and

GERD [192-197]. In FD the benefit of acid suppressive drugs is less clear. Studies examining the

efficacy of acid suppressive agents in this condition are difficult to evaluate [153,157,198-203],

but about half of the studies examining H2RA’s show a modest positive effect. Only a few

randomised-placebo controlled trials studied the efficacy of PPI’s in FD and showed a benefit of

about 10% over placebo [137-140,204]. Taken together with the well known large placebo effect

of any drug in FD [153,157], these data explain that a trial of treatment with an acid-suppressive

agent reduces symptoms in a fair number of patients with uninvestigated dyspepsia. Nevertheless,

several objections can be made to such an approach. The benefit of an attempt to treat the patient

empirically depends on the number of patients becoming permanently symptom-free afterwards or

at least for a long period. Unfortunately, in patients with PUD and GERD, symptoms usually recur

after stopping the treatment [61,193,205,206]. It is very likely that the same can be said about

many patients with FD, as this is also often a chronic condition with the majority of the patients

still experiencing symptoms after one year [7,15,207-209]. Therefore, it can be anticipated that,

unless one is willing to continue acid suppression without a definite diagnosis, a trial with such

medication just postpones endoscopy in most patients. This assumption is supported by the study

of Bytzer et al. [131] who showed that 67% of dyspeptic patients who received empirical

treatment with H2RA’s underwent endoscopy within a year. The second objection that can be

made against this approach is a practical one. If endoscopy is performed after all, signs of PUD or

GERD may be obscured if the medication has not been withdrawn for a sufficiently long period

before endoscopy as commonly occurs in clinical practice. A trial with a prokinetic drug

(cisapride) will be of benefit in patients with GERD and probably in some with PUD and FD and

will also have a considerable placebo effect in FD patients [153,157]. Therefore, it can be

anticipated that, like acid suppression, a trial of such treatment will have a favourable response in a

fair number of patients. Most drawbacks mentioned above for acid suppression, however, are also

pertinent for this class of drugs, be it that prokinetic agents are probably less likely to obscure

endoscopic signs of GERD and PUD. The recently described cardiac side effects of cisapride will

limit its use now.

Chapter 2

28

What about continuing treatment without a definite diagnosis? Although this is an option,

several objections can be made to such an approach. First, patients with PUD will be effectively

treated, but a far more cost-effective treatment (H. pylori eradication) is to be preferred [210].

Second, if patients with GERD, who are likely to respond well to potent acid suppression, are not

subjected to endoscopy, Barrett's metaplasia will not be detected. This condition occurs in about

10 to 15% of all patients with GERD [211-213] and is associated with an increased risk for

oesophageal cancer [214-216], be it that it was recently suggested that this relative risk has been

exaggerated [217]. Recent studies have indeed confirmed the latter [218,219]. Whether or not

failure to diagnose Barrett’s oesophagus is significant depends on one’s view on the necessity of

endoscopic surveillance of these patients. This is still an unsettled issue [214-216,220-226].

Finally, it has been argued that empirical treatment with acid-suppressive agents may lead to a

significant delay in diagnosing malignant disease [227]. In the western world, however, this

contention does not seem to hold as gastrointestinal malignancy is extremely rare in dyspeptic

patients below 45-50 years if alarm symptoms are not present [228-230].

If one chooses to test for H. pylori one should first have knowledge of the reliability of the test

results (its positive and negative predictive value). This is not only dependent on the characteristics

of the test (sensitivity and specificity) but also on the prevalence of the infection in the population.

For instance, as the prevalence of H. pylori infection is decreasing in the younger age group , the

likelihood of a false positive test in this age group increases. This may be acceptable if a “test-and-

scope” strategy is followed as the test can be confirmed by one or more biopsy-based methods, but

it is certainly not acceptable if one follows a “test-and-treat” strategy. In that case, only highly

accurate tests (with very high specificity) are acceptable and serology will soon not be suitable

anymore. Urea breath tests [231,232] and stool antigen tests [233-235] may be valuable

alternatives.

Screening for H. pylori will provide one of two possibilities: a positive test result or a negative

test result. A negative test result leads to the same approach in both the “test-and-scope” and “test-

and-treat” strategies. In such a patient PUD is highly unlikely, leaving FD and GERD as the most

likely diagnoses. In the absence of alarm symptoms malignancies are extremely rare under the age

of 50 [228-230]. Most patients can, therefore, safely be treated blindly with acid-suppression.

Endoscopy will not change the management in most of these patients [177]. In case of a positive

test result the “test-and-scope” strategy will detect most patients with PUD. Anti-H. pylori


29

treatment can be limited to these patients as the benefit of such a treatment is only shown

decisively in PUD [236]. Treatment can be guided by susceptibilty testing based on H. pylori

culture. In the “test-and-treat” strategy, however, all H. pylori-positive patients receive anti-H.

pylori treatment. This approach will be beneficial in most patients with PUD and possibly a few

patients with FD, but in the large majority of patients there will be no short-term benefit.

Nevertheless, two additional arguments support the eradication of H. pylori in all patients with

uninvestigated dyspepsia. First, in contrast to all other therapeutic options, anti-H. pylori treatment

only takes 1 to 2 weeks and, if successful, eliminates a potential cause of dyspepsia for lifetime, as

re-infection is rare in the Western world [69,237-242]. Second, it may have a positive effect by

preventing future disease, especially PUD [71-73]. Whether gastric cancer is prevented by

eradication of H. pylori [243] is still highly uncertain but some have advocated prophylactic anti-

H. pylori treatment for patients on long term acid-suppressive therapy as these patients may be at

increased risk of developing gastric cancer [244-246]. This issue is still debated [247,248] but it

may support H. pylori eradication in dyspeptic patients.

On the other hand, several arguments have been cited against treatment of the infection in all

patients with uninvestigated dyspepsia in whom H. pylori is detected. First of all, it will not

diminish symptoms in the large majority of patients and just expose them to the side effects of the

anti-H. pylori regimen [249-253] even though these are usually mild and in a trial setting form a

reason to discontinue treatment in less than 5% of all patients [250-254].

Second, treating H. pylori infection in all these patients may induce antibiotic resistance. To

what extent this occurs in clinical practice is not known. Antibiotic resistance in H. pylori is

indeed a major problem [254-258] and its prevalence seems to be increasing [259,260]. Whether

this increase is caused by anti-H. pylori treatment or by the prescription of antibiotics for other

indications, however, is not known. As the current anti-H. pylori regimens are so effective, with

eradication rates of > 95% in susceptible strains [255], induction of resistance should be a rare

event, as long as the regimen is properly prescribed and time is taken to instruct and motivate the

patient [261]. Third, eradication of H. pylori may induce other diseases, especially GERD

[124,125,262] but this does not seem to be a clinically significant problem in the majority of

patients [110,263]. Considering all these arguments favouring and renouncing H. pylori treatment

in uninvestigated dyspepsia (table 6), it is the authors’ view that the weight of the evidence favours

treatment of the infection when it is diagnosed.

Chapter 2

30

Table 6.

Arguments favouring and renouncing Helicobacter pylori treatment in uninvestigated

dyspepsia

• favouring anti-H. pylori treatment

• a single short-term treatment, with life-long effects when beneficial

• prevents future disease (PUD, gastric cancer)

• renouncing anti-H. pylori treatment

• no benefit in most patients

• exposing patients to side-effects

• induction of anti-biotic resistance (H. pylori and other gastrointestinal bacteria)

• possible induction of GERD after H. pylori eradication

Conclusions and recommendations

It is unlikely that a single strategy will be applicable in all dyspeptic patients in primary care.

Nevertheless, in our view, the “test-and-treat” strategy is currently a rational way to deal with

many patients with uncomplicated dyspepsia, provided that the following conditions are met. First,

the positive predictive value of the test should be adequate. Therefore, an accurate test should be

used and the prevalence of the infection should be monitored. Second, an effective anti-H. pylori

treatment regimen should be used and time should be taken to instruct the patient in order to

improve compliance. If the prevalence of H. pylori gets low, as is the case in the younger age

group in western countries, the “test-and-treat” strategy becomes less suitable. “Test-and-scope”

becomes more appropriate as the positive non-invasive H. pylori test can be confirmed by two or

more biopsy based tests. In our view, if the infection is confirmed, it should be treated even if no

PUD is detected as such an approach may prevent future diagnostic dilemmas and possibly future

disease. In both strategies all patients testing negative for H. pylori can safely be treated with acid

suppression (or prokinetics if symptomatology suggests a motility disorder). If this treatment is

successful, it should be tried to gradually stop the medication. If this approach fails and the patient

remains symptomatic endoscopy should be considered mainly to reassure the patient, as it is

unlikely to reveal underlying disease or change treatment. If the prevalence of H. pylori gets very

low as is likely to be the case in the future in western societies, non-invasive testing for the micro-

organism is probably not opportune any more. In that situation empirical treatment may be the way


31

to proceed even though it may just postpone endoscopy in quite some patients. Acid-suppressive

drugs, especially PPI’s, are then the drugs of choice. Prokinetics may become an alternative but

cisapride has recently been withdrawn from the market and other comparably effective prokinetic

drugs, both in patients with FD and GERD, are not yet available. In some patients, however, the

approach should be individualized as dictated by their personal history. Any alarm symtom should

urge for an immediate endoscopy. A strong family history for peptic ulcer disease may be a

persuasive argument in favour of a test and treat approach even if the prevalence of H. pylori is

low. If fear of cancer is deep-rooted early endoscopy should be considered. Therefore, the taking

of an adequate medical history remains the mainstay of the approach of every patient. This holds

no less for the dyspeptic patient in primary care.

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70-1.

51

Chapter 3

The approach of dyspepsia in primary care. A randomised trial

comparing “test-and-treat” with prompt endoscopy.

Nicolaas L. A. Arents 1, Jacob C. Thijs 2, Anton A. van Zwet 1, Marco Oudkerk Pool 3,

Jan-Mark Götz 4, Ger Th. van de Werf 5, Klaas Reenders 5, Wim J. Sluiter 6, and Jan H.

Kleibeuker 7

1 Regional Public Health Laboratory, Groningen / Drenthe2 Department of Gastroenterology, Bethesda Hospital, Hoogeveen3 Department of Gastroenterology, Isala Clinics, Zwolle4 Department of Gastroenterology, Wilhelmina Hospital, Assen5 Department of General Practice, University Hospital, Groningen6 Department of Pathology, University Hospital, Groningen7 Department of Gastroenterology, University Hospital, Groningen

in press, Archives of Internal Medicine.

Chapter 3

52

Abstract

Background

The clinical value of the “test-and-treat” strategy in the approach of dyspepsia has only been

evaluated in a few secondary care studies. The majority of the dyspeptic patients, however, are

managed by their primary care physician only. This study evaluated the “test-and-treat” strategy in

primary care.

Methods

Patients consulting their general practitioner (GP) for dyspepsia were randomised to either

direct open-access endoscopy with H. pylori testing or a “test-and-treat” strategy based on H.

pylori serology. All patients received eradication therapy if H. pylori positive. In the 12 months

follow-up period, any additional treatment or referral for further investigations was left at the

discretion of the GP. At the end of the study, data were collected in both study groups concerning

the number of endoscopies performed, changes in symptom severity, changes in quality of life,

patient satisfaction, and the use of medical resources.

Results

Two hundred seventy patients were enrolled (129 endoscopy and 141 “test-and-treat” patients).

The prevalence of H. pylori infection was 38% and 37% in the “test-and-treat” and endoscopy

group respectively. In the “test-and-treat” group, forty-six patients (33%) were referred for

endoscopy during follow-up. Improvement in symptom severity, quality of life, and patient

satisfaction was comparable in both groups. Patients in the “test-and-treat” group paid more

dyspepsia related visits to their GP (p=0.005). Patients in the endoscopy group were more often

prescribed PPI’s (p=0.007), whereas patients in the “test-and-treat” group were more often

prescribed prokinetic drugs (p=0.005).

Conclusions

The “test-and-treat” strategy proved to be as effective and safe as prompt endoscopy. Only a

minority of patients were referred for endoscopy after “test-and-treat”.

A randomised trial comparing “test-and-treat” with prompt endoscopy

53

Introduction

In primary care, general practitioners (GP’s) usually treat younger dyspeptic patients

empirically with acid suppressing agents before considering endoscopy. This approach, in which

endoscopy is reserved for patients with persisting symptoms, was asserted to be the most cost-

effective strategy by the American College of Physicians in 1985 [1]. Since then, however, new

data have become available, suggesting that this common practice may have to be reconsidered.

First of all, the most common cause of dyspepsia in general practice is functional dyspepsia [2]

and there is no sound evidence to support the use of acid suppression in this condition [3,4].

Secondly, although peptic ulcer disease (PUD) and gastro-oesophageal reflux disease (GERD) are

initially adequately treated by a course of acid suppression, they usually relapse after

discontinuation of the drug [5-8]. Therefore, in most of these patients endoscopy is merely

postponed when the aforementioned approach is followed [9]. Finally, eradication of Helicobacter

pylori (H. pylori) in PUD patients harbouring this bacterium is far more cost-effective than

symptomatic acid suppressive treatment [10].

These data have led to the development of several alternative strategies. Some authors

proposed a strategy based on non-invasive screening for H. pylori and referring only those patients

testing positive for endoscopy (“test-and-scope”) [11,12]. Others have modified this approach and

recommended a “test-and-treat” strategy, in which patients testing positive receive anti-H. pylori

treatment, possibly obviating the need for endoscopy [13-15]. On the other hand, it has also been

suggested that immediate endoscopy may be the most cost-effective approach [16,17].

All strategies have been extensively tested by decision-analytic studies [18-25], but only a few

studies have actually compared the different approaches in a clinical setting [9,13-15,26,27]. These

clinical studies, however, suffer from one significant drawback. None of them was performed

entirely in a primary care setting, where most patients with dyspepsia are treated [28,29]. This

article presents the results of a study comparing a “test-and-treat” strategy to prompt endoscopy.

The study was performed entirely in a primary care setting, using open-access endoscopy facilities.

Methods

According to the Rome I criteria, dyspepsia was defined as persistent or recurrent pain or

discomfort centred in the upper abdomen [30]. Patients consulting any of the 56 participating

general practitioners (GP’s) for dyspepsia were eligible for the study if the GP considered the

Chapter 3

54

symptoms severe enough to warrant endoscopy or the prescription of acid-suppressive medication.

The GP’s were instructed not to include patients with symptoms suggestive of GERD (heartburn

or acid regurgitation as the only or predominant symptom). Other exclusion criteria were age

younger than 18 years; the presence of sinister symptoms (including the first appearance of

dyspeptic symptoms after the age of 55); previously documented PUD or GERD; previous surgery

of the upper alimentary tract, other than uncomplicated cholecystectomy; previous anti-H. pylori

treatment; the use of proton pump inhibitors (PPI’s), bismuth compounds, or antibiotics in the

month before inclusion; the use of non-steroidal anti-inflammatory drugs (NSAID’s) other than

low-dose acetyl salicylic acid in the preceding two weeks; known allergy for the drugs used in the

study; suspected poor compliance; pregnancy or lactation; and participation in any other study.

Patients were withdrawn from the study if any malignancy was diagnosed or if the patient got

pregnant. After giving written informed consent, patients were randomised by their GP.

Randomisation was stratified for each GP by supplying each GP with blocks of four sealed

envelopes, ensuring two patients randomised to the “test-and-treat” and two patients to the

endoscopy group.

In the endoscopy group the patients were referred for open-access endoscopy, which was

performed within 2 weeks after inclusion. At endoscopy three biopsy samples were taken within 2

cm of the pylorus for H. pylori culture (n=1) and histology (haematoxylin-eosin staining and either

Giemsa staining or immuno staining for H. pylori) (n=2). Antibiotic susceptibility for

metronidazole (resistant if Minimal Inhibitory Concentration (MIC) ≥ 8) and claritromycin

(resistant if MIC ≥ 2) was tested by E-test (AB-Biodisk, Sweden). A venous blood sample was

drawn for the detection of anti-H. pylori IgG antibodies using a commercially available assay

(Pyloriset EIA-G III, Orion Diagnostica, Espoo, Finland). This assay has shown a sensitivity and

specificity of 98.4% and 88.4% respectively in a previous study performed in the same region

[31]. A peptic ulcer was defined as a mucosal break of > 5 mm with a clearly visible ulcer crater in

either the stomach or the duodenal bulb. Erosive inflammation of the duodenal bulb was

considered to be an appearance of PUD. If at endoscopy PUD was detected, the patient was

prescribed ranitidine immediately after endoscopy. Patients with reflux oesophagitis were

prescribed lansoprazole for a period of three months. The dosages were left at the discretion of the

endoscopist. In all other cases the patients were referred back to their GP awaiting culture and

histology results. All H. pylori infected patients (either culture or histology positive) received a


55

one-week anti-H. pylori regimen guided by susceptibility testing: lansoprazole (30 mg bid),

amoxicillin (1000 mg bid), and either metronidazole (500 mg bid) (LAM) if metronidazole-

sensitive or clarithromycin (500 mg bid) (LAC) if metronidazole-resistant. If susceptibility was

not tested the microorganism was considered metronidazole-resistant. In a previous study,

performed in the same region [32], resistance of H. pylori to clarithromycin was shown to be very

rare (about 2%). If a patient had normal endoscopic findings and was H. pylori negative, cisapride

(20 mg bid) was prescribed for four weeks by the GP according to protocol. Peptic ulcers, erosive

gastritis, and erosive duodenitis in the absence of H. pylori infection were treated after confering

with the gastroenterologist, usually with acid suppressive agents.

In the “test-and-treat” group, a venous blood sample was drawn for H. pylori serology. If

testing positive the patients were prescribed LAC by the GP. If negative, the GPs were encouraged

to prescribe cisapride (20 mg bid) for four weeks. After the initial treatment according to protocol,

the GP was free to manage the patients according to his or her own insights. This could include

either a change in treatment, referral for diagnostic tests, including endoscopy, or referral to a

gastroenterologist or another specialist.

The patients were followed for 12 months. At inclusion and at 12 months, the patients were

asked to complete a dyspepsia questionnaire evaluating the severity of eight dyspeptic symptoms

(pain in the upper abdomen, bloating, nausea, burping / belching, flatulence, heartburn, sour taste,

and halitosis) on a 5-point Likert scale [33]. Quality of life (QoL) was evaluated at inclusion and

after 12 months using the RAND-36 questionnaire, a validated Dutch translation of the SF-36

questionnaire [34,35]. At 12 months, patients were invited to donate a second blood sample for H.

pylori serology. Successfull H. pylori treatment was defined as a decrease in titer of IgG H. pylori

antibodies ≥ 40% [36]. For the detection of a decrease in antibody level both the entry sample and

the 12 months sample were examined in the same run. Patient's satisfaction with medical treatment

was evaluated on a 5-point Likert scale. Whenever a patient did not return a questionnaire, they

were contacted by telephone by one of the investigators (NLAA) and urged to do so for up to three

times.

At the end of the study, the GPs' records of each patient (electronic and/or patient charts) were

reviewed by a single investigator (NLAA). Data were collected concerning the number of visits for

dyspepsia, the number of visits for other reasons, additional investigations (endoscopy, abdominal

ultrasound, and X-rays of the gastrointestinal tract), specialist referrals, and hospital admissions.

Chapter 3

56

Finally, all pharmacies were asked to provide a printed list of all medications delivered to the

patients within the one-year follow-up period. The dyspepsia-related use of medical resources was

calculated for medical consultations, dyspepsia-related investigations, hospital admissions, and

dyspepsia-related prescriptions.

Differences in mean scores and mean changes in scores between groups were compared after

correction for continuity by the method of Yates and Cochran. Mean changes in score within a

group were evaluated after correction for continuity by the method of Pitman. Frequencies were

compared by the chi-square test with Yates' correction. Differences in continuous variables

between groups were studied by the Mann-Whitney-U-test. All tests were two-sided with a level of

significance of p < 0.05.

The study protocol was approved by the medical ethical committee of the University Hospital

Groningen.

Results

Two hundred and eighty one patients consented to participate in the study (Figure 1). Eleven of

these patients were not enrolled for the following reasons: failed to return the first questionnaires

(n=2), no show-up at endoscopy (n=4), no biopsies taken at endoscopy (n=2), suspected poor

compliance (alcohol abuse (n=1), shortly staying foreigner (n=1)), presence of sinister symptoms

(n=1). Therefore, 270 patients were enrolled, 141 patients in the “test-and-treat” group and 129 in

the endoscopy group. Quality of life, time elapsed since the first appearance of symptoms as well

as the duration of current symptoms were not significantly different between both groups (p

ranging between 0.21 and 0.98 for all items). The mean score of one symptom in the dyspepsia

questionnaire (nausea) was significantly higher in the “test-and-treat” group (2.09 vs 1.79,

p=0.039). Mean scores of the other symptoms, however, as well as other baseline characteristics

(table 1) were comparable.

Forty-six patients (32.6%) randomised to the “test-and-treat” group (16 serologically H. pylori-

positive) were referred for endoscopy during follow-up. One endoscopy was aborted due to lack of

co-operation of the patient. The endoscopic diagnoses in the remaining 45 patients as well as in the

patients randomised to the endoscopy group are summarised in figure 2. The endoscopic diagnosis

most commonly made in both groups was GERD (“test-and-treat” group: 31.1%; endoscopy

group: 28.7%). GERD included oesophageal erosions (Savary-Miller grade 1 and higher) and/or


57

Figure 1.

Flow diagram of the study patients.

281 patients consented to

participate

270 patients randomised

11 patients excluded

141 assigned to

“test-and-treat”

129 assigned to

prompt endoscopy

124 patients included

in final analysis

120 patients included

in final analysis

follow-up data not

available in 17 patients

follow-up data not

available in 9 patients

Chapter 3

58

Table 1.

Baseline characteristics of patients in the “test-and-treat” group and endoscopy group.

“Test-and-treat” group

(n=141)

Endoscopy group

(n=129) p-value

% %

Median age 47 44 0.35

Male 70 49.6 59 45.7 0.62

Smokers 55 39.0 47 36.4 0.75

Alcohol use (>1 unit daily) 24 17.0 22 17.1 0.87

Born outside The Netherlands 9 6.4 6 4.7 0.72

H. pylori positive 54 38.3 48 37.2 0.95

Barrett’s metaplasia (n=2). Duodenal ulcer disease (including erosive duodenitis in four) was

found in one of the 46 patient in the “test-and-treat” group and in 10 patients in the endoscopy

group (7.8%; one patient also had GERD). GU was not found in the “test-and-treat” group and in

one patient (0.8%; this patient also had GERD) in the endoscopy group. Erosive gastritis was

found in one (2.2%) and three (2.3%) patients in the “test-and-treat” and endoscopy group

respectively. In three patients with PUD both histology and culture were negative for H. pylori.

One of these patients (“test-and-treat” group) had been treated for H. pylori infection, but still

showed erosive inflammation of the duodenal bulb. The other two patients, randomised to the

endoscopy group, both had a duodenal ulcer. In both serology was negative as well. The H. pylori

status in all endoscoped patients is shown in table 2. In one 54 year old patient, randomised to the

“test-and-treat” group a gastric malignancywas diagnosed three weeks after inclusion. Further

examination showed multiple liver metastases with ascites and the patient passed away within 6

months. In both study groups, endoscopy did not show any abnormality or just a hiatal hernia in

most patients (62.2% and 60.5% in the “test-and-treat” and endoscopy group respectively).

Statistical analysis did not show any significant difference in frequencies of endoscopic diagnoses

between both study groups (p=0.83). When compared to the biopsy based detection methods in the

endoscopy group (H. pylori positive when histology and/or culture positive), the serological test

showed a sensitivity, specificity, positive and negative predictive value of 86.7% (95% confidence

interval (CI) 73.2 - 95.0), 92.5% (95% CI 84.4 - 97.2), 86.7% (95% CI 73.2 - 95.0), and 92.5%


59

Figure 2.

Endoscopic diagnoses in the “test-and-treat” patients referred for endoscopy (n=45) and the

endoscopy group (n=129).

Test-and-treat group

Endoscopy group

N = normal or hiatal hernia only; GERD = Gastro-oesophageal reflux disease; EG = Erosive

gastritis; DU = Duodenal ulcer (including erosive duodenitis); GU = Gastric cancer

GERD31,1%

EG2,2%

GC2,2%

DU2,2%

N62,2%

N60,5%

DU7,8%

EG2,3%

GU0,8%

GERD28,7%

Chapter 3

60

Table 2.

H. pylori status in patients in the endoscopy group and in endoscoped patients in the

“test-and-treat” group.

Endoscopic diagnoses Endoscopy group (n=129) “Test-and-treat” group (n=45)

H. pylori positive1 H. pylori positive2

% %

GERD 12/37 32.4 3/14 21.4

DU 8/10 80.0 1/1 100.0

GU 1/1 100.0 0/0 0

Erosive gastritis 1/3 33.3 0/0 0

Gastric malignancy 0/0 0 1/1 100.0

normal 26/78 33.3 11/28 39.3

GERD = Gastro-oesophageal reflux disease; DU = Duodenal ulcer (including erosive duodenitis);

GU = Gastric ulcer. 1as determined by culture and/or histology; 2as determined by serology at

inclusion

(95% CI 84.4 - 97.2) respectively.

Follow-up data were not available of 17 patients in the “test-and-treat” group (12.1%) and of 9

patients in the endoscopy group (7.0%) (p=0.22) for the following reasons: failed to return follow-

up questionnaires (n=22), pregnancy (n=2) or malignancy (n=2) diagnosed during follow-up.

Besides the patient with gastric cancer mentioned above, Hodgkin’s disease was diagnosed in

another patient. Baseline characteristics (table 1) and mean symptom severity score of these

dropout patients were comparable to the baseline characteristics of the remaining patients.

Symptom changes in both study groups were evaluated by subtracting the scores in the last

questionnaire from the scores in the first questionnaire for each symptom. Figure 3 shows the

mean change for each symptom in both groups. All symptoms improved over the study period and

improvement was comparable in both study groups.

The scores of the QoL categories evaluated by the RAND-36 questionnaire (change in health,

general health perception, bodily pain, energy / fatigue, general mental health, role limitations due

to personal or emotional problems, role limitations due to physical health problems, social

functioning, and physical functioning) were calculated according to the instructions of the


61

questionnaire's designers [34]. To detect any improvement or worsening within a category, the

first QoL scores were subtracted from the 12-month QoL scores. The median score changes for all

categories are shown in figure 4. There was an improvement in most categories in both groups and

the differences between the two groups were not statistically significant.

The use of medical resources in each study group is summarised in table 3. The mean number

of dyspepsia-related GP visits was significantly higher in the “test-and-treat” group (p=0.005). In

the endoscopy group, more patients were prescribed PPIs (p=0.007), making the mean standard

daily dosage (SDD) in this group higher than in the “test-and-treat” group. The mean SDD in the

patients receiving PPIs, however, was comparable in both groups (p=0.71). Sixty eight percent of

the patients in the endoscopy group who were prescribed PPIs had endoscopy proven GERD.

Prokinetic drugs were significantly more often prescribed in the “test-and-treat” group (p=0.005),

making the mean SDD in this group higher than in the endoscopy group. The use of H2RA and

antacids in both study groups was comparable. In both groups, the same number of patients did not

receive any medication for dyspepsia (18.5% and 23.3% for the “test-and-treat” and endoscopy

group respectively (p=0.5). Apart from endoscopy, the number of dyspepsia related medical

investigations were comparable in both groups (p=0.34), as was the mean number of specialist

referrals for dyspepsia (p=0.81). Just one patient (“test-and-treat” group) was admitted to hospital

because of upper abdominal symptoms. The use of medical resources for reasons other than

dyspepsia was comparable in both groups.

A second serum sample was available of 83/96 (86.5%) H. pylori-positive patients. According

to our definition anti-H. pylori treatment had been succesfull in 39/45 (86.7%) patients in the “test-

and-treat” group and 34/38 (89.5%) in the endoscopy group (p=0.95).

Patient satisfaction with medical treatment in both groups is shown in figure 5. The large

majority of patients (94.4%) were satisfied and there were no differences between both study

groups (p=0.75).

Chapter 3

62

Figure 3.

Mean change in symptom score in the test-and-treat” (n=124) and the endoscopy group

(n=120) and the mean difference between both scores with the 95% confidence interval.

Figure 4.

Median change in Quality of life scores in the “test-and-treat” (n=124) and the endoscopy

group (n= 120).

-20% -10% 0% 10% 20% 30% 40% 50% 60% 70%

Test-and-treat Endoscopy

-0,5 -0,3 -0,1 0,1 0,3 0,5 0,7 0,9 1,1 1,3 1,5

Endoscopy Test-and-treat Difference between Test-and-treat and Endoscopy


63

Figure 5.

Patient satisfaction in the “test-and-treat” (n=124) and endoscopy group (n= 120).

0%

10%

20%

30%

40%

50%

60%

VeryUnsatisfied

Unsatisfied ModeratelySatisfied

Satisfied VerySatisfied

Test-and-treat Endoscopy

Chapter 3

64


65

Chapter 3

66

Discussion

This study showed that prompt endoscopy in patients consulting their primary care doctor for

dyspepsia led to similar symptom resolution, improvement in quality of life and patient

satisfaction as compared to the “test-and-treat” strategy. Over two thirds of the patients treated

according to the “test-and-treat” strategy were not referred for endoscopy during a follow-up

period of one year. Our study is the first study comparing the “test-and-treat” strategy to prompt

endoscopy in a primary care setting. As most dyspeptic patients are entirely managed by their

general practitioner (GP), the “test-and-treat” approach should be evaluated in that setting. Lassen

et al. recently compared the “test-and-treat” strategy to prompt endoscopy [15]. In that study,

however, follow-up was performed completely in a secondary care setting, which may have biased

the results. Reassurance and treatment by a gastroenterologist with a special interest in dyspepsia

may be more effective than that by a GP, resulting in a decreased use of medical resources and a

higher patient satisfaction. Our primary care study has several other positive features. After the

initial investigation and treatment guidelines, GPs were allowed to treat and manage their patients

according to their own insights. Therefore, it is to be assumed that the outcome of the “test-and-

treat” strategy in our study will be very similar to the outcome if a similar approach would be

followed in daily clinical practice, thus increasing the external validity of the results. Secondly, all

questionnaires regarding symptoms, quality of life, and patient satisfaction were self administered,

avoiding any interpretation bias by the GP. Finally, anti-H. pylori treatment was prescribed to all

H. pylori infected patients. Although the benefit of anti-H. pylori treatment has only been

demonstrated unequivocally in patients with PUD, we aimed to prevent any bias by a possible

effect in patients with FD or GERD. Inevitably, our study had some drawbacks. Patients were

recruited by their own GP which may have introduced a potential selection bias, limiting the

comparability between the study patients and the normal dyspeptic population. Moreover, our

study does not allow a comparison of the “test-and-treat” strategy to the currently most often

chosen approach (empiric treatment). We chose to include a control group (prompt endoscopy)

rather than an empiric study group, in order to determine whether the “test-and-treat” strategy

missed any important disease.The presence of abnormalities in the “test-and-treat” group at

inclusion are assumingly similar to the endoscopic findings in the prompt endoscopy group. It

should be noted that in the endoscoped “test-and-treat” patients H. pylori positive PUD was not

diagnosed. This strongly indicates that the “test-and-treat” strategy correctly identified all patients


67

with H. pylori associated PUD and that these patients received adequate treatment. The only

patient in the “test-and-treat” group in whom PUD was diagnosed, had erosive inflammation in the

duodenal bulb at endoscopy six months after anti-H. pylori treatment. Endoscopic biopsies

excluded H. pylori infection. NSAIDs were not prescribed to this patient during the study year, but

over the counter use can not be excluded.

The efficacy of the anti-H. pylori regimen was > 85% in both study arms. Susceptibility testing

prior to anti-H. pylori treatment did not seem to improve treatment succes rate, but this may be

related to the low prevalence of clarithromycin resistance in our region. An often cited argument

against the “test-and-treat” strategy is the possibility of overlooking gastric or oesophageal cancer.

In our study, gastric cancer was diagnosed in one patient three weeks after inclusion, which is a

duration of doctors delay that has been suggested to be acceptable [37].

PPIs were more often prescribed in the endoscopy group than in the “test-and-treat” group.

Most likely, this was related to the prescription of PPIs in nearly all patients with endoscopy

proven GERD, as recommended by the study guidelines, whereas in primary care it is common

practice to start treatment with a H2RA if GERD is expected in uninvestigated patients.

Prokinetics were more often prescribed in the “test-and-treat” group, which was clearly the direct

result of our study protocol advising prokinetics in all non-invasively tested H. pylori negative

patients. Other dyspepsia related drugs were equally used in both study groups. Patients in the

“test-and-treat” group more often consulted their GP for dyspepsia, which may indicate more

effective treatment or better reassurance in the endoscopy group. The use of other medical

resources, including specialist referrals, hospital admissions, and additional investigations for

dyspepsia other than endoscopy were comparable in both study groups.

Most current guidelines advocate empiric treatment with acid-suppressive agents as a first

approach to dyspepsia. Whether the “test-and-treat” strategy is more cost-effective than an empiric

approach can not be concluded from this study. One of the most important variables, however,

influencing cost-effectiveness of any approach for dyspepsia is the number (and cost) of

endoscopies [18,19]. The study by Bytzer et al, comparing an empiric acid-suppressive approach

with H2RA with prompt endoscopy, showed a 34% reduction in the number of endoscopies in the

first year [9]. When compared to this outcome, both our study and the study by Lassen et al.

showed a higher reduction in number of endoscopies (67.4% and 60% respectively). It could

therefore be hypothesized that the “test-and-treat” approach may be more cost-effective than

Chapter 3

68

empiric acid suppression. It should be emphasized, however, that the cost-effectiveness of a “test-

and-treat” strategy largely depends on the prevalence of PUD and H. pylori infection in the

population. The reported declining prevalence of H. pylori has a negative impact on the predictive

value of non-invasive tests for H. pylori. Moreover, the decreasing prevalence of PUD will limit

the number of patients most clearly benefiting from anti-H. pylori treatment.

In conclusion, a “test-and-treat” approach of dyspeptic patients in primary care is as effective

and safe as a strategy in which all patients are immediately referred for endoscopy. Only a

minority (33%) of patients are referred for endoscopy after “test-and-treat”.

Acknowledgements

The SENSE study (Strategy : Endoscopy versus Serology) was supported by a grant from Aventis

Pharma, Hoevelaken, The Netherlands.

References

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Public Policy Committee, American College of Physicians, Philadelphia, Pennsylvania. Annals of




3. Talley NJ. Drug treatment of functional dyspepsia. Scandinavian Journal of Gastroenterology 1991;

182: 47-60.

4. Veldhuyzen van Zanten SJ, Cleary C, Talley NJ, Peterson TC, Nyren O, Bradley LA et al. Drug

treatment of functional dyspepsia: a systematic analysis of trial methodology with recommendations for

design of future trials. American Journal of Gastroenterology 1996; 91: 660-673.

5. Graham DY, Colon-Pagan J, Morse RS, Johnson TL, Walsh JH, McCullough AJ et al. Ulcer

recurrence following duodenal ulcer healing with omeprazole, ranitidine, or placebo: a double-blind,

multicenter, 6-month study. The Omeprazole Duodenal Ulcer Study Group. Gastroenterology 1992; 102:

1289-1294.

6. Dammann HG, Walter TA. Efficacy of continuous therapy for peptic ulcer in controlled clinical trials.

Alimentary Pharmacology & Therapeutics 1993; 7 (suppl 2): 17-25.

7. Hetzel DJ, Dent J, Reed WD, Narielvala FM, Mackinnon M, McCarthy JH et al. Healing and

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8. Koelz HR, Birchler R, Bretholz A, Bron B, Capitaine Y, Delmore G et al. Healing and relapse of

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9. Bytzer P, Hansen JM, Schaffalitzky de Muckadell OB. Empirical H2-blocker therapy or prompt

endoscopy in management of dyspepsia. Lancet 1994; 343: 811-816.

10. Unge P, Jonsson B, Stalhammar NO. The cost effectiveness of Helicobacter pylori eradication versus

maintenance and episodic treatment in duodenal ulcer patients in Sweden. Pharmacoeconomics 1995; 8:

410-427.

11. Sobala GM, Crabtree JE, Pentith JA, Rathbone BJ, Shallcross TM, Wyatt JI et al. Screening




13. Heaney A, Collins JSA, Watson RGP, McFarland RJ, Bamford KB, Tham TCK. A prospective

randomised trial of a test-and-treat policy versus endoscopy based management in young Helicobacter

pylori positive patients with ulcer-like dyspepsia, referred to a hospital clinic. Gut 1999; 45: 186-190.

14. Jones R, Tait C, Sladen G, Weston-Baker J. A trial of a test-and-treat strategy for Helicobacter pylori

positive dyspeptic patients in general practice. International Journal of Clincial Practice 1999; 53: 413-

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2000; 356: 455-460.

16. Bytzer P. Diagnosing dyspepsia: any controversies left ? Gastroenterology 1996; 110: 302-306.

17. Tytgat G, Hungin APS, Malfertheiner P, Talley N, Hongo M, McColl K et al. Decision-making in

dyspepsia: controversies in primary and secondary care. European Journal of Gatroenterology &

Hepatology 1999; 11: 223-230.

18. Fendrick AM, Chernew ME, Hirth RA, Bloom BS . Alternative management strategies for patients

with suspected peptic ulcer disease. Annals of Internal Medicine 1995; 123: 260-268.

19. Silverstein MD, Petterson T, Talley NJ. Initial endoscopy or empirical therapy with or without testing

for Helicobacter pylori for dyspepsia: a decision analysis. Gastroenterology 1996; 110: 72-83.

20. Sonnenberg A. Cost-benefit analysis of testing for Helicobacter pylori in dyspeptic subjects. American


21. Briggs AH, Sculpher MJ, Logan RP, Aldous J, Ramsay ME, Baron JH. Cost effectiveness of

screening for and eradication of Helicobacter pylori in management of dyspeptic patients under 45 years

of age. British Medical Journal 1996; 312: 1321-1325.

22. Ebell MH, Warbasse L, Brenner C. Evaluation of the dyspeptic patient: a cost-utility study. Journal of

Family Practice 1997; 44: 545-555.

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23. Ofman JJ, Etchason J, Fullerton S, Kahn KL, Soll AH. Management strategies for Helicobacter


Medicine 1997; 126: 280-291.

24. Laheij RJ, Severens JL, Jansen JB, van de Lisdonk EH, Verbeek AL. Management in general

practice of patients with persistent dyspepsia. A decision analysis. Journal of Clinical Gastroenterology

1997; 25: 563-567.

25. Olson AD, Deutsch D . Combined empiric treatment and specific Hp screening and treatment decrease

the cost of evaluating epigastric abdominal pain. Gastroenterology 1998; 114: A248.

26. Laheij RJ, Severens JL, van de Lisdonk EH, Verbeek AL, Jansen JB. Randomized controlled trial of

omeprazole or endoscopy in patients with persistent dyspepsia: a cost-effectiveness analysis. Alimentary

Pharmacology & Therapeutics 1998; 12: 1249-1256.

27. Duggan A, Elliot C., Tolley K, Whynes D, Hawkey CJ, Logan RF. Randomised controlled trial of

four dyspepsia management strategies in primary care with 12 months follow-up. Gastroenterology

2000; 118: A438.

28. Jones R, Lydeard S . Prevalence of symptoms of dyspepsia in the community. British Medical Journal

1989; 298: 30-32.

29. Muris JWM, Starmans R, Fijten GH, Crebolder HFJM, Krebber TFWA, Knottnerus JA.


30. Talley NJ, Colin-Jones D, Koch KL, Nyren O, Stanghellini V. Functional dyspepsia: a classification

with guidelines for diagnosis and management. Gastroenterology International 1991; 4: 145-160.

31. Thijs JC, van Zwet AA, Thijs WJ, Oey HB, Karrenbeld A, Stellaard F et al. Diagnostic tests for

Helicobacter pylori: a prospective evaluation of their accuracy, without selecting a single test as the gold

standard. American Journal of Gastroenterology 1996; 91: 2125-2129.

32. Arents NLA, van Zwet AA, Thijs JC, Degener JE, Kleibeuker JH. A six-year surveillance of H.

pylori resistance in a large study population in the Netherlands (n=5946): Prevalence of clarithromycin

resistance increases, while metronidazole resistance does not increase. Gastroenterology 2001; 120:

A229.

33. Veldhuyzen van Zanten SJO, Tytgat KMAJ, Pollak PT, Goldie J, Goodacre RL, Riddell RH et al.

Can severity of symptoms be used as an outcome measure in trials of non-ulcer dyspepsia and

Helicobacter pylori associated gastritis ? J Clin Epidemiol 1993; 46: 273-279.

34. van der Zee KI, Sanderman R, Heyink J. De psychometrische kwaliteiten van de MOS 36-item Short

Form Health Survey (SF-36). T Soc Gezondheidszorg 1993; 71: 183-191.

35. Ware JE, Sherbourne CD. The RAND-36 Short-form Health Status Survey: 1. Conceptual framework

and item selection . Medical Care 1992; 30: 473-481.


71

36. Kosunen TU, Seppala K, Sarna S, Sipponen P. Diagnostic value of decreasing IgG, IgA, and IgM

antibody titres after eradication of Helicobacter pylori. Lancet 1992; 339: 893-895.

37. Anonymous. American Gastroenterology Association medical position paper: evaluation of dyspepsia.


73

Chapter 4

The accuracy of the Helicobacter pylori Stool Antigen test (HpSA

test) in diagnosing Helicobacter pylori in treated and untreated

patients.

Nicolaas L. A. Arents 1, Anton A. van Zwet 1, Jacob C. Thijs 2, Albertine de Jong 1,

Marco Oudkerk Pool 3, and Jan H. Kleibeuker 4.

1 Regional Public Health Laboratory, Groningen / Drenthe2 Department of Internal Medicine, Bethesda Hospital, Hoogeveen3 Department of Gastroenterology, Isala Clinics, Zwolle4 Department of Gastroenterology, University Hospital, Groningen

European Journal of Gastroenterology & Hepatology 2001;13:383-386.

Chapter 4

74

Summary

Objective and design

To evaluate the performance of the HpSA test in detecting H. pylori infection and monitoring the

effect of treatment. This was done in two separate studies using either a biopsy- or 13C-urea breath

test based gold standard (in untreated and treated patients respectively).

Setting

Endoscopy units of two general hospitals.

Patients

Hundred twenty-eight dyspeptic patients undergoing endoscopy in the first study. Sixty-five

patients receiving anti-H. pylori treatment in the second study.

Results

Sensitivity and specificity in untreated patients were 96.3% and 81.8% respectively. Seven days

after treatment these figures were 20% and 95% and four weeks after treatment 40% and 95%

Conclusion

The HpsA test is accurate in untreated patients but fails in monitoring treatment success.

The accuracy of the Helicobacter pylori Stool Antigen test

75

Introduction

The optimal diagnostic approach in patients with dyspepsia is still controversial. Upper

endoscopy is frequently performed as the primary diagnostic test, but it is costly and in most

patients no underlying disease can be identified [1]. It has been suggested that a strategy based on

non-invasive testing for Helicobacter pylori (H. pylori) could be more cost-effective. Such a

strategy could either imply the referral of only H. pylori-positive patients for endoscopy (“test and

scope” strategy) [2-5] or subjecting H. pylori-positive patients to anti-H. pylori treatment (“test

and treat” strategy) [6-8]. Whatever approach is chosen, its efficacy is highly dependent on the

accuracy of the test used to diagnose H. pylori infection. So far, two non-invasive diagnostic tests

for H. pylori were clinically available: ELISA based serology and urea breath tests using either 13C

or 14C. Both tests have been thoroughly validated [9-17] but have their limitations. The urea breath

tests are expensive and the samples need to be examined in a specialised laboratory. ELISA

serology may still be positive several years after (accidental) eradication of H. pylori by antibiotic

treatment, as antibody titres decline very slowly [18,19]. Recently, a new easy to perform non-

invasive diagnostic test has been introduced: the H. pylori Stool Antigen test (HpSA test, Meridian

Diagnostics, Cincinnati, OH, USA), based on the detection of H. pylori antigens in faeces. The

first studies evaluating this test are highly promising [20,21]. Moreover, the test was claimed to be

suitable for early monitoring of treatment success [21,22]. In that respect the test could be

competitive with breath testing. In this study we evaluated the HpSA test in a group of untreated

patients and also tested its performance to monitor treatment success.

Methods

In the first part of our study (study A), we evaluated the accuracy of the HpSA test in patients

who had never been treated for H. pylori infection. Patients undergoing gastroduodenoscopy in

two regional hospitals in the Netherlands were invited to bring their faeces the day after

endoscopy. Patients using protonpump inhibitors (PPI) and/or antibiotics during the previous four

weeks were excluded. At endoscopy, four biopsy specimens were taken within 4 cm of the

pylorus: one for culture, two for histology (HE and Giemsa stain), and one for rapid in-house

urease testing. Culture and rapid urease testing were performed as described previously [23]. A

patient was considered H. pylori infected if culture was positive and/or histology and rapid urease

test were both positive. All other combinations were regarded as H. pylori negative.

Chapter 4

76

In the second part of our study (study B), we evaluated the HpSA test after treatment. Culture

proven H. pylori positive patients receiving anti H. pylori therapy were asked to collect a faecal

sample 7 days and 4 weeks after cessation of treatment. Success of treatment was determined by

UBT 4 and 8 weeks after the end of therapy. Patients were not allowed to use PPI's or antibiotics

in the time between treatment and the second UBT. If both UBT tests were negative, the patient

was considered H. pylori negative. If any UBT was positive, endoscopy was performed and H.

pylori status was determined in the same way as in study A. UBT was performed using 100 mg13C-urea and orange juice as a test meal. The test was considered positive if δ 13CO2/12CO2 was ≥ 5

per mil at 20 and/or 30 minutes after intake of the labelled urea. The HpSA test was performed

according to the manufacturer's guidelines by a single experienced technologist in batches of 48

samples including two controls.

Optical density (OD) was determined by dual wavelength spectrophotometry (450/630 NM).

Cut-off values advocated by the manufacturer were used: negative < 0.1; equivocal ≥ 0.1 but <

0.12; positive ≥ 0.12. We also drew a Receiver-Operator Characteristic curve (ROC-curve) and

recalculated the optimal cut-off value for our population. All patients gave informed consent and

the local ethics committee approved the study protocol.

Results

Hundred twenty eight patients participated in study A (54 males, age range 16-84 years, mean

53). Fifty-five of them (43%) were H. pylori positive by gold standard. Using the manufacturer’s

cut-off values, eight patients (6.2%) had an equivocal HpSA test result (one patient H. pylori-

positive and seven H. pylori negative by gold standard). The HpSA test identified 52 (96.3%) of

the remaining 54 H. pylori positive patients correctly. Fifty-four (81.8%) of the remaining 66 H.

pylori-negative patients were negative by HpSA test. The ROC-curve is shown in figure 1. The

optimal cut-off in our population proved to be OD = 0.2. This cut-off value led to a sensitivity and

specificity of 89.1% and 89.0% respectively.

Sixty-five patients were included in study B (29 males, mean age 58, range 18-76). In five

patients (7.7%) UBT was positive after treatment (first UBT in four and second in one) and

persisting H. pylori infection was confirmed by endoscopy in all of them. In this study no


77

Figure 1.

Receiver operator characteristic curve based on data from study A.

equivocal HpSA results were encountered using the manufacturer’s cut-off values. Using these

values, the HpSA test, performed 7 days after cessation of treatment was positive in only one

(20.0%) of the five patients in whom treatment failed. Four weeks after cessation of treatment this

figure was 2 out of five (40.0%). In the 60 patients who were successfully treated, HpSA was

falsely positive in three both 7 days and 4 weeks after cessation of treatment (specificity at both

occasions 95.0%). Figures changed only marginally when the optimal cut-off value determined by

the ROC-curve obtained from study A was applied (after 7 days sensitivity 20.0% and specificity

96.7%; after 4 weeks sensitivity 20.0% and specificity 98,3%). The data are summarised in table 1.

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0,900

1,000

0,000 0,200 0,400 0,600 0,800 1,000

sens

itivi

ty

1 - specificity→ →

↑

Chapter 4

78


79

Discussion

The good performance of the HpSA test in study A is in agreement with the data reported by

Vaira et al. [20]. The accuracy was somewhat lower in our study, but due to the limited number of

patients tested, confidence intervals are rather wide and our results still suggest that in untreated

patients, the HpSA test may be a valuable diagnostic method. When compared to the UBT, the test

has got the advantage that it does not need a specialised laboratory and is less expensive. In our

view, however, it has still to be shown that the HpSA test has a significant advantage above the

cheaper serological test for screening purposes.

It has been claimed that the HpSA test can be used for monitoring treatment success and that

successful eradication of H. pylori can be confirmed as early as 1 week after treatment [24,25]. In

that respect the HpSA test could have an important advantage over serological tests as antibody

titres decline slowly and it takes about six months before serology can reliably be used to confirm

treatment success [17,19]. Moreover, in contrast with serological monitoring of treatment success

for which pre- and post treatment titres are needed, only one faecal sample is required when the

HpSA test is used for that purpose. Several authors found the HpSA test to have a sensitivity and

specificity of > 91% and > 95% respectively in treated patients 4 weeks after cessation of therapy

[21,22,26]. Our study, however, does not substantiate these findings as sensitivity and positive

predictive value were poor both 7 days and 4 weeks after cessation of therapy. The employment of

the most optimal cut-off value for our population, as determined in study A, did not improve the

accuracy of the HpSA test in treated patients.

The difference between our results and the excellent results reported by others is striking and

we have no adequate explanation for that difference. It is difficult to conceive that differences

between the populations studied could be an explanation and it is also unlikely to be due to

methodological problems, as the HpSA test is very easy to perform. Nevertheless, our results led

us to question our methodology and we asked independent investigators to repeat the tests on the

same post-treatment faecal samples. The results, however, did not change.

Based on our results, we have to conclude that the HpSA test is accurate in detecting the

presence of H. pylori in untreated patients. In our hands, however, the HpSA test failed as a

method to monitor treatment success.

Chapter 4

80

References





3. Sobala GM, Crabtree JE , Pentith JA, Rathbone BJ, Shallcross TM, Wyatt JI et al. Screening


4. Mendall MA, Goggin PM, Marrero JM, Molineaux N, Levy J, Badve S et al. Role of

Helicobacter pylori serology in screening prior to endoscopy. European Journal of Gatroenterology &

Hepatology 1992; 4: 713-717.

5. Slade PE, avidson AR, teel A, ox RA, lackburn PA. Reducing the endoscopic workload: does

serological testing for Helicobacter pylori help ? European Journal of Gatroenterology & Hepatology

1999; 11: 857-862.

6. Ofman JJ, Etchason J, Fullerton S, Kahn KL, Soll AH. Management strategies for Helicobacter


Medicine 1997; 126: 280-291.

7. Breslin NP, Lee J, Buckley M, O'Morain C. Screening for Helicobacter pylori in young dyspeptic

patients referred for investigation--endoscopy for those who test negative. Alimentary Pharmacology

& Therapeutics 1998; 12: 577-582.

8. Moayyedi P, Zilles A, Clough M, Hemingbrough E, Chalmers DM, Axon ATR. The effectiveness

of screening and treating Helicobacter pylori in the management of dyspepsia. European Journal of


9. Graham DY, Klein PD, Evans DJ, Jr., Evans DG, Alpert LC, Opekun AR et al. Campylobacter

pylori detected noninvasively by the 13C-urea breath test. Lancet 1987; 1: 1174-1177.

10. Logan RPH. The 13C-UBT for the detection of Helicobacter pylori. In: Rathbone BJ, Heatly RV,

editors. Helicobacter pylori and gastroduodenal disease. London: Blackwell Scientific Publications,

1992: 88-107.

11. Thijs JC, van Zwet AA , Thijs WJ, Oey HB, Karrenbeld A, Stellaard F et al. Diagnostic tests for

Helicobacter pylori: a prospective evaluation of their accuracy, without selecting a single test as the

gold standard. American Journal of Gastroenterology 1996; 91: 2125-2129.

12. Meijer BC, Thijs JC, Kleibeuker JH, van Zwet AA, Berrelkamp RJ . Evaluation of eight enzyme

immunoassays for detection of immunoglobulin G against Helicobacter pylori. Journal of Clinical

Microbiology 1997; 35: 292-294.


81

13. Perez-Perez GI, Dworkin BM, Chodos JE, Blaser MJ. Campylobacter pylori antibodies in humans.

Annals of Internal Medicine 1988; 109: 11-17.

14. Talley NJ, Newell DG, Ormand JE, Carpenter HA, Wilson WR, Zinsmeister AR et al.

Serodiagnosis of Helicobacter pylori: comparison of enzyme-linked immunosorbent assays. Journal of

Clinical Microbiology 1991; 29: 1635-1639.

15. Crabtree JE, Shallcross TM, Heatley RV, Wyatt JI. Evaluation of a commercial ELISA for

serodiagnosis of Helicobacter pylori infection. J Clin Pathol 1991; 44: 326-328.

16. Cullen DJ, Cullen KJ, Collins BJ, Christiansen KJ, Epis J. Serological assessment of

Helicobacter pylori eradication [letter]. Lancet 1992; 340: 1161-1162.

17. Cutler A, Schubert A, Schubert T. Role of Helicobacter pylori serology in evaluating treatment

success. Digestive Diseases and Science 1993; 38: 2262-2266.

18. Cutler AF, Prasad VM. Long-term follow-up of Helicobacter pylori serology after successful

eradication. American Journal of Gastroenterology 1996; 91: 85-88.

19. Thijs JC, van Zwet AA , Meijer BC, Berrelkamp RJP. Serology to monitor the efficacy of anti-

Helicobacter pylori treatment. European Journal of Gatroenterology & Hepatology 1994; 6: 579-583.

20. Vaira D, Malfertheiner P, Megraud F, Axon ATR, Deltenre M, Hirschl AM et al. Diagnosis of

Helicobacter pylori infection with a new non-invasive antigen-based assay. Lancet 1999; 354: 30-33.

21. Braden B, Teuber G, Dietrich CF, Caspary WF, Lembcke B. Comparison of new faecal antigen

test with 13C-urea breath test for Helicobacter pylori infection and monitoring eradication treatment :

prospective clinical evaluation. British Medical Journal 2000; 320: 16-17.

22. Vaira D, Malfertheiner P, Megraud F, Axon ATR, Deltenre M, Gasbarrini G et al. Noninvasive

antigen-based assay for assessing Helicobacter pylori eradication: a European multicenter study.


23. van Zwet AA, Thijs JC , Kooistra-Smid AMD, Schirm J, Snijder JAM. Sensitivity of culture

compared with that of polymerase chain reaction for detection of Helicobacter pylori from antral

biopsy samples. Journal of Clinical Microbiology 1993; 31: 1918-1920.

24. Van 't Hoff BWM, van der Ende A, van der Hulst RWM, Roorda PLH, Houben MHMG,

Rauws EAJ et al. Helicobacter pylori antigen stool specimen as a possible monitoring tool for

eradication therapy. Gut 1998; 43 (suppl 2): A49 (abstract).

25. Bleau BL, Veldhuyzen van Zanten S, Best L, Hutchison D, Blevins J, Thee D. Determination of

eradication of Helicobacter pylori (Hp) by the Hp stool antigen test (HPSAT). Gut 1998; 43 (suppl 2):

A50 (abstract).

26. Oderda G, Rapa A, Ronchi B, Lerro P, Pastore M, Staiano A et al. Detection of Helicobacter

pylori in stool specimens by non-invasive antigen enzyme immunoassay in children: multicentre

Italian study. British Medical Journal 2000; 320: 347-348.

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82

27. Braden B, Teuber G, Dietrich CF, Caspary WF, Lembcke B. Comparison of new faecal antigen

test with 13C-urea breath test for Helicobacter pylori infection and monitoring eradication treatment :

prospective clinical evaluation. British Medical Journal 2000; 320: 16-17.

83

Chapter 5

Does the declining prevalence of Helicobacter pylori unmask

patients with idiopathic peptic ulcer disease ?

Trends over an eight-year period.

Nicolaas L. A. Arents 1, Jacob C. Thijs 2, Anton A. van Zwet 1, and

Jan H. Kleibeuker 3.

1 Regional Public Health Laboratory, Groningen / Drenthe

2 Department of Gastroenterology, Bethesda Hospital, Hoogeveen

3 Department of Gastroenterology, University Hospital, Groningen


Chapter 5

84

Abstract

Objectives

Recent North American studies have suggested that the prevalence of Helicobacter pylor (H.

pylori) infection in patients with peptic ulcer disease (PUD) not using non-steroidal anti-

inflammatory drugs (NSAIDs) has been overestimated. The decreasing prevalence of H. pylori

could lead to a relative increase in the number of patiens with this idiopathic PUD (IPUD).

Aim

This study aimed to investigate the prevalence of IPUD and any possible trends.

Methods

The reports of all upper gastro-intestinal endoscopies performed in a Dutch regional hospital over

the period 1991 to 1998 were reviewed. If a gastric and/or duodenal ulcer had been diagnosed,

data concerning a possible H. pylori infection (culture, histology, rapid in-house urease test) were

retrieved. If H. pylori tests were negative, hospital files were examined for possible NSAIDs use

or other rare causes of PUD. When these were not found, stored biopsy specimens were tested for

H heilmanii by PCR.

Results

PUD was diagnosed in 405 endoscopies (GU=159, DU=235, GU+UD=11). In 349 of these cases

(86.2%), H. pylori infection was found. Thirty three of the 56 H. pylori negative patients used

NSAIDs and three patients had Crohn’s disease leaving 20 patients with IPUD (4.9%, GU=12,

DU=8). Timetrends over the study period showed a decrease of H. pylori associated PUD

(p<0.002) and an increase of NSAIDs associated PUD (p<0.0005). The prevalence of IPUD

remained stable (p=0.978).

Conclusion

IPUD is rare in the Netherlands and its prevalence did not increase over a period of eight years.

The prevalence of H. pylori associated PUD is decreasing while the prevalence of NSAIDs

associated PUD is increasing.

Does the declining prevalence of Helicobacter pylori unmask patients with IPUD ?

85

Introduction

Peptic ulcer disease (PUD) has been strongly associated with Helicobacter pylori (H. pylori)

infection [1]. It has previously been suggested that about 80% of the patients with gastric ulcer

(GU) and over 90% of the patients with duodenal ulcer (DU) are infected with this bacterium

[2,3]. Most PUD patients, not infected with H. pylori, were thought to have used non-steroidal

anti-inflammatory drugs (NSAIDs) [2,4,5]. In a small minority of patients, however, neither H.

pylori nor NSAIDs can be incriminated, even after exclusion of falsely negative tests for H. pylori

(mainly due to recent use of acid-suppressive drugs or antibiotics [6,7]) or concealed use of

NSAIDs [8]. If rare causes of the ulcer (table 1) are not identified, these patients are considered to

suffer from idiopathic PUD (IPUD).

Table 1.

Rare causes of Duodenal and Gastric ulcer disease

Helicobacter heilmanii (Gastrospirillum hominis)

Crohn’s disease

Penetrating carcinoma / lymphoma's

Gastrinoma

Cytomegalovirus infection

Tuberculosis

Ischaemia

It has recently been suggested, however, that the prevalence of H. pylori in patients with

NSAID-negative DU has been overestimated and the combined results of several large well-

performed North American studies showed that no more than 75% of those patients are infected

with H. pylori [9]. Two other recent North American studies also reported lower prevalence rates

of 74% and 61% respectively [10,11]. Another argument supporting the suggestion that the

significance of H. pylori in PUD has been overestimated, comes from studies reporting that

duodenal ulcers still recur in about 20% of the patients after successful H. pylori eradication [5,12-

14]. These observations have resulted in the hypothesis that in some patients with PUD H. pylori

infection actually masks the presence of IPUD [7,15]. If this is true, it can be anticipated that the

decreasing prevalence of H. pylori infection will reveal an increasing number of patients with

Chapter 5

86

IPUD. This would have significant implications for the approach of patients with peptic ulcer

disease in the near future.

On the other hand, most of these s tudies were performed in North America and recent studies

from other parts of the world still have shown H. pylori prevalence rates to exceed 90% in DU

[3,15-19]. The aim of this retrospective study was to investigate the prevalence of IPUD in an

European population and to detect any change in that prevalence over a period of 8 years.

Methods

The study was performed in a small regional hospital in the north-eastern part of The

Netherlands, serving a population of 75.000 persons. The reports of all upper gastrointestinal

endoscopies performed over an 8-year period (1991-1998) were studied. After exclusion of the

endoscopy reports of patients with previous gastric resection and patients undergoing endoscopy

for the follow up of previously diagnosed disease, endoscopy reports describing a gastric or

duodenal ulcer (defined as a mucosal break of > 5 mm with a clearly visible ulcer crater) were

selected. In the large majority of these PUD patients biopsy samples had been taken for the

diagnosis of H. pylori infection by rapid urease testing, histology (Haematoxylin-eosin and

Giemsa stain), and H. pylori culture as previously described [20]. The patients were classified as

having “H. pylori associated PUD” if any of these tests was positive and as having “H. pylori-

negative PUD” if no test was positive and at least two tests were negative. Endoscopy reports were

omitted from further evaluation, if the patient was not tested for H. pylori, if the test results were

lost, or if only one test was performed and described as negative.

In case of “H. pylori-negative PUD” the hospital files of the patients were retrieved and the

clinical presentation was noted. Moreover, the files were carefully searched for documented

NSAID use, especially in the presence of diseases (such as arthritis and cardiovascular disease)

making the use of NSAIDs or (low-dose) aspirin likely. If NSAID use could be demonstrated the

“H. pylori-negative PUD” was subclassified as “NSAID-associated PUD”. The hospital files were

also searched for the possible presence or developement of other rare causes of PUD mentioned in

table 1. If such a cause was not identified and both H. pylori and NSAID use could not be

incriminated paraffin embedded antral biopsy specimens were retrieved and assessed by a nested

PCR procedure for the presence of Helicobacter heilmanii (H. heilmanii). The biopsy specimens

were extracted from the paraffin and DNA isolation was performed on each entire biopsy


87

specimen according to Boom et al. [21]. Subsequently, all bacterial 16S rDNA-genes were

amplified by PCR using the following primers: forward 5’ CTTTACGCCATTTAATCCG 3’ and

reversed 5’ AGAGTTTGATCCTGGTTCAG 3’. PCR conditions were 40 cycles of 30 sec at 95ºC,

30 at 60ºC, and 60 at 72ºC, followed by a final extension of 10 minutes at 72ºC. The amplification

products were used in a second PCR amplification to detect the presence of H. heilmanni using the

following primers: forward 5’ AACGATGAAGCTTCTAGCTTGCTAG 3’ and reverse 5’

GTGCTTATTCGTTAGATACC-GTCAT 3’. In this case, PCR conditions were 35 cycles of 30

sec at 95ºC, 30 at 50ºC, and 60 at 72ºC followed by a final extension of 10 minutes at 72ºC. The

presence of a specific amplificate for H. heilmanii was visualised by standard 2% agarose gel

electrophoresis and ethidium bromide staining.

Group proportions were compared by Chi-square testing with Yates' correction. The presence

of time trends was investigated by Cochran's linear trend analysis. A p value of <0.05 was

considered significant.

Results

In the period from 1991 to 1998 6241 endoscopies of the upper alimentary tract were

performed. Of these, 1381 endoscopies were performed in patients with previous gastric resection

or for the follow up of previously diagnosed disease, leaving 4860 endoscopy reports for

evaluation. In 439 of these reports (9.0%) peptic ulcer was described ((GU in 173 (39.4%), DU in

254 patients (57.9%), and GU+DU in 12 patients (2.7%)). Thirty-four of the 439 endoscopy

reports (7.7%) were omitted as the H. pylori status could not be determined adequately. Three

hundred-forty-nine (86.2%) of the remaining 405 reports described at least one positive H. pylori

test result and the ulcers were therefore classified as “H. pylori associated PUD”. During the study

period, the percentage of PUD that was associated with H. pylori decreased significantly

(p=0.002). In 56 reports (13.8%) “H. pylori-negative PUD” was documented, which proved to be

“NSAID associated PUD” in 33 cases (8.1%). The proportion of PUD patients having “NSAID

associated PUD” increased significantly over the study period (p<0.0005). Finally, in 23 cases

(5.7%) neither H. pylori infection nor NSAID use could be incriminated. Three of these patients

suffered from Crohn’s disease (one patient with GU, one patient with DU, and one patient with

both GU and DU), leaving 20 patients with IPUD (4.9%). PCR on H. heilmanii was negative in all

of them. The proportion of PUD patients having IPUD did not change significantly over the study

Chapter 5

88

period (p=0.978) and varied between 0% and 12.5% (mean 4.9%). Table 2 summarises the

absolute numbers in the different subgroups. Figure 1 shows the proportions of the subgroups

within the PUD group, omitting the three patients with Crohn's disease.

the three patients with Crohn's disease.

Table 2.

Absolute numbers of H. pylori associated PUD, NSAID associated PUD, Crohn’s disease

associated PUD, and IPUD over the study period.

Year ‘91 ‘92 ‘93 ‘94 ‘95 ‘96 ‘97 ‘98 Total

Hp+ 39 54 49 52 34 42 40 39 349

Hp- / NSAID+ 1 2 1 3 4 8 5 9 33

Hp- / NSAID- / M. Crohn + 0 0 1 0 1 0 0 1 3

Hp- / NSAID- / M. Crohn - (IPUD) 3 2 1 3 3 3 5 0 20

total PUD 43 58 52 58 42 53 50 49 405

Hp = Helicobacter pylori, PUD = peptic ulcer disease, IPUD = idiopathic peptic ulcer disease,

NSAID = non-steroidal anti-inflammatory drugs

When GU and DU were considered separately (GU+DU omitted because of the limited number

of patients), it was shown that the relative proportions of both conditions within the total PUD

group did not change significantly during the study period (p=0.2 and p=0.6 respectively). H.

pylori infection was more prevalent in DU (mean 89.4%) than in GU (mean 81.1%) (p=0.03), but

its prevalence in DU decreased significantly during the study period (p=0.01), whereas the

prevalence of H. pylori in GU decreased only marginally (p=0.052). In both groups, NSAID use

could be demonstrated in an increasing number of patients (p=0.023 for GU and p=0.003 for DU).

GU seemed to represent IPUD more often than DU (7.5% vs 3.4%) but the difference did not reach

statistical significance (p=0.10) and no significant trends were observed within either group during

the study period. In a remarkably high percentage of patients with IPUD (50%) the first clinical

presentation proved to be an upper gastro-intestinal haemorrhage.


89

Figure 1.

The contribution of all PUD subgroups: H. pylori associated PUD, NSAID associated PUD,

and IPUD to the total number of patients with PUD.

Discussion

In accordance with many other studies [2,3], our study showed that the prevalence of H. pylori

infection is higher in DU (89.4%) than in GU (81.1%). It also showed that the prevalence of H.

pylori in patients with DU visiting a Dutch hospital is gradually decreasing, even though most

patients still harbor the infection. The prevalence of H. pylori in patients with GU seemed to

decrease, but the trend did not reach statistical significance. Most patients with “H. pylori–

negative PUD” had used NSAIDs as documented in their endoscopy reports and hospital files. The

proportion of PUD that could reasonably be ascribed to NSAID use increased significantly from

1991 to 1998. This was true for both DU and GU. The proportion of PUD patients in whom

neither H. pylori nor NSAID use could be incriminated did not increase significantly during the

study period. In none of these patients H. heilmanii could be demonstrated by PCR but in three of

them Crohn’s disease was detected. The proportion of PUD patients having IPUD was only 4.9%.

Being retrospective, our study has some inevitable shortcomings. First of all, even though any

NSAID use is usually mentioned in the endoscopy report and the hospital files were thoroughly

checked for it, it cannot be excluded that some patients with IPUD actually had concealed NSAID

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

91 92 93 94 95 96 97 98

H. pylori + H. pylori- / NSAID + True IPUD

Chapter 5

90

use. Secondly, it could be argued that the prevalence of H. pylori infection was underestimated as

the tests for H. pylori might have been falsely negative due to sampling error or the recent use of

antibiotics or proton pump inhibitors. As we usually take both antral and corpus biopsy samples

and test by three methods (culture, histology and rapid urease test) we consider this less likely.

Moreover, we excluded patients if only one test result was available and described as negative. In

any case, if either possibility would be true it would only strengthen our conclusion that IPUD is

still rare in The Netherlands.

The low prevalence of IPUD in our study is in strong contrast with data from the USA [9-11] but

in line with prevalences found in other European studies [17,18]. An explanation for the observed

difference between Europe and the USA is yet to be given, as differences in detection methods for

H. pylori are an unlikely explanation and concealed NSAID use was carefully excluded in the

American studies [9-11].

When reviewing the hospital files of patients with IPUD we were struck by the observation that

50% of the patients acutely presented with an upper gastro-intestinal haemorrhage. To our

knowledge, this has not previously been discribed. This is remarkable as it has been estimated that

this complication occurs in approximately 15% of all PUD patients [22].

In conclusion, in a Dutch population the proportion of PUD associated with H. pylori infection

is gradually decreasing. This decrease is accompanied by an increase in the proportion of PUD

attributable to NSAID use. These developments are especially seen in DU patients and less so in

GU. The proportion of PUD that cannot be attributed to either H. pylori or NSAID use (IPUD)

seems to remain stable and is still very small. In those patients H. heilmanii does not seem to be a

significant cause but other diseases, especially Crohn’s disease should be looked for.

References

1. Marshall BJ. Helicobacter pylori. American Journal of Gastroenterology 1994; 89: S116-S127.



3. Nishikawa K, Sugiyama T, Kato M, Ishizuka J, Komatsu Y, Kagaya H et al. Non-Helicobacter

pylori and non-NSAID peptic ulcer disease in the Japanese population. European Journal of


4. Borody TJ, Brandl S, Andrews P, Jankiewicz E, Ostapowicz N. Helicobacter pylori-negative

gastric ulcer. American Journal of Gastroenterology 1992; 87: 1403-1406.


91

5. Lanas A, Remacha B, Sainz R, Hirschowitz BI. Study of outcome after targeted intervention for

peptic ulcer resistant to acid suppresion therapy. American Journal of Gastroenterology 2000; 95:

513-519.

6. Nensey YM, Schubert TT , Bologna SD, Ma CK. Helicobacter pylori-negative duodenal ulcer. Am

J Med 1991; 91: 15-18.

7. Howden CW. What causes Helicobacter pylori negative non-NSAID-related ulcers ? In: Hunt RH,

Tytgat GNJ, editors. Helicobacter pylori. Basic Mechanisms to clinical cure 2000. Dordrecht: Kluwer

Academic Publishers, 2000: 339-345.

8. Hirschowitz BI, Lanas A. Intractable upper gastrointestinal ulceration due to aspirin in patients who

have undergone surgery for peptic ulcer. Gastroenterology 1998; 114: 883-892.

9. Ciociola AA, McSorley DJ, Turner K, Sykes D, Palmer JBD. Helicobacter pylori infection rates in

duodenal ulcer patients in the United States may be lower than previously estimated. American


10. Peterson WL, Ciociola AA, Sykes DL, McSorley DJ, Webb DD, The RBC H.pylori study group.

Ranitidine bismuth citrate plus clarithromycin is effective for healing duodenal ulcers, eradicating

Helicobacter pylori and reducing ulcer recurrence. Alimentary Pharmacology & Therapeutics 1996;

10: 251-261.

11. Jyotheeswaran S, Shah AN, Jin HO, Potter GD, Ona FV, Chey WY. Prevalence of Helicobacter

pylori in peptic ulcer patients in greater Rochester, NY: is empirical triple therapy justified ?


12. Lanza F, Ciociola AA, Sykes D, Heath A, McSorley DJ, Webb PM. Ranitidine bismuth citrate plus

clarithromycin is effective in eradicating H. pylori, and preventing ulcer relapse. Gastroenterology

1996; 91: 917.

13. Sprung DJ. The natural history of duodenal ulcer disease and how it relates to Helicobacter pylori.

American Journal of Gastroenterology 1997; 92: 1655.

14. Laine L, Hopkins RJ, Girardi LS . Has the impact of Helicobacter pylori therapy on ulcer

recurrence in the United States been overstated? A meta-analysis of rigorously designed trials.





16. Cutler AF, Schubert TT. Patient factors affecting Helicobacter pylori eradication with triple therapy.


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17. Gisbert JP, Blanco M, Mateos JM, Fernandez-Salazar L, Fernandez-Bermejo M, Cantero J et

al. H. pylori-negative duodenal ulcer prevalence and causes in 774 patients. Digestive Diseases and

Science 1999; 44: 2295-2302.

18. McColl KEL, El-Nujumi AM, Chittajallu RS, Dahill SW, Dorrian CA, El-Omar E et al. A study

of the pathogenesis of Helicobacter pylori negative chronic duodenal ulcer. Gut 1993; 34: 762-768.

19. Pan ZJ, Berg DE, van der Hulst R, Su WW, Raudonikiene A, Xiao SD et al. Prevalence of

vacuolating cytotoxin production and distribution of distinct vacA alleles in Helicobacter pylori from

China. Journal of Infectious Diseases 1998; 178: 220-226.

20. Thijs JC, van Zwet AA , Thijs WJ, Oey HB, Karrenbeld A, Stellaard F et al. Diagnostic tests for

Helicobacter pylori: a prospective evaluation of their accuracy, without selecting a single test as the

gold standard. American Journal of Gastroenterology 1996; 91: 2125-2129.

21. Boom R, Sol CJA, Heijtink R, Wertheim-van Dillen PME, van der Noordaa J. Rapid purification

of hepatitis B virus DNA from serum. Journal of Clinical Microbiology 1991; 29: 1804-1811.

22. Del Valle J, Cohen H, Laine L, Scheiman JM. Acid peptic disorders. In: Yamada Y, editor.

Textbook of Gatroenterology. Philadelphia, New York, Baltimore: Lippincott, Williams & Wilkins,

1999: 2023-2045.

93

Chapter 6

The influence of changing prevalences in metronidazole and

clarithromycin resistance on the efficacy of Helicobacter pylori

treatment.

Nicolaas L. A. Arents 1, Anton A. van Zwet 1, Jacob C. Thijs 2, John E. Degener 3, and

Jan H. Kleibeuker 4.

1 Regional Public Health Laboratory, Groningen / Drenthe2 Department of Internal Medicine, Bethesda Hospital, Hoogeveen3 Department of Medical Microbiology, University Hospital, Groningen4 Department of Gastroenterology, University Hospital, Groningen


Chapter 6

94

Abstract

Dyspeptic patients managed by the “test-and-treat” strategy in primary care receive “blind”

anti-H. pylori therapy as knowledge about antimicrobial susceptibility is lacking. Antibiotic

resistance in H. pylori, however, has a significant impact on the efficacy of the frequently used

triple regimens.

The aim of this study was to detect trends in the prevalence of metronidazole and

clarithromycin resistance in biopsy specimens obtained at endoscopy of more than 16000 patients

collected during an eight year period. Moreover we have calculated the theoretical efficacy of two

triple regimens (PAM: protonpump inhibitor - amoxicillin - metronidazole, and PAC: protonpump

inhibitor - amoxicillin - clarithromycin) when prescribed “blindly” to 100 consecutive H. pylori

positive dyspeptic patients based on data from meta-analysis studies in the literature.

During the study period, the prevalence of metronidazole resistance decreased significantly

from 28.4% in 1996 to 13.5% in 2001. The prevalence of clarithromycin resistance remained

stable at about 2.2%. Both triple regimens had an overall efficacy of > 80%, but the PAM regimen

was more effective than the PAC regimen and will become increasingly more effective when the

prevalence of metronidazole resistance continues to decline.

Introduction

The majority of the world population is infected with Helicobacter pylori (H. pylori) [1]. This

bacterium is the most important cause of gastritis and peptic ulcer disease (PUD) and is also

associated with the development of gastric cancer [2-4]. Nevertheless, most patients harbouring H.

pylori will never develop any symptoms [5], and therefore, most guidelines have recommended to

treat the infection only in PUD patients as only those patients will most likely benefit from H.

pylori eradication. To diagnose PUD, however, endoscopy is needed and an increasing number of

younger dyspeptic patients is managed by their general practitioner (GP) by the “test-and-treat”

approach [6]. In this strategy, dyspeptic patients are screened for H. pylori by non-invasive means

and those testing positive receive anti-H. pylori therapy without prior endoscopy. H. pylori

negative patients are usually reassured or treated empirically with acid-suppressive or prokinetic

agents [7,8]. This approach is likely to be more cost-effective, as it leads to a decrease in the

number of endoscopies and has therefore been embraced by several international authorities in the

The influence of changing prevalences of antibiotic resistance in Helicobacter pylori

95

field of gastroenterology [9-12]. In view of these developments, most H. pylori infections will be

treated without prior knowledge of antibiotic susceptibility.

Treating H. pylori infection in primary care is only possible when short, effective, and well

tolerated, regimens are available which are easy to comply with. The most commonly prescribed

anti-H. pylori regimens in primary care are the triple regimens which fulfil most of these

conditions. These 7-14-day regimens contain a proton-pump inhibitor, amoxicillin, and either

metronidazole (PAM) or clarithromycin (PAC). According to the recommendations of the

European Society of Primary Care Gastroenterology, treatment of H. pylori infection in primary

care should achieve a cure rate of at least 80% [13]. Such a cure rate can easily be achieved by

both triple regimens [14], provided that the patient is carrying a metronidazole and/or

clarithromycin susceptible strain [15]. In the presence of a metronidazole and/or clarithromycin

resistance, however, the efficacy of these triple regimens is significantly lowered [14,15].

Resistance to amoxicillin is extremely rare and so-far only one case of a stable amoxicillin

resistant H. pylori strain has been reported [16]. Widespread resistance to metronidazole and

clarithromycin in H. pylori would, therefore, seriously hamper the suitability of the “test-and-treat”

strategy and recently, several studies have suggested that the prevalence of metronidazole and

clarithromycin resistance may be increasing [17-20]. Even though most of these studies were

performed in a relatively small number of strains it can be questioned whether the goal of the

European Society of Primary Care Gastroenterology will remain achievable in the near future if

the PAM or PAC regimens are used. The aim of this study was to determine the prevalence of

metronidazole and clarithromycin resistance in a large population over an eight-year period and to

investigate the presence of trends. Moreover, we calculated the theoretical efficacy of the PAM

and PAC regimen when prescribed “blindly” at different prevalence rates of antibiotic resistance.

Methods

The study was performed in the northeastern part of the Netherlands in the microbiology

laboratories of five different regional hospitals (Refaja Hospital Stadskanaal, Bethesda Hospital

Hoogeveen, Diaconessen Hospital Meppel, Scheper Hospital Emmen and Regional Hospital

Hardenberg). In those laboratories H. pylori cultures have been performed by the same

standardised protocol since 1995. Briefly, biopsy specimens obtained at endoscopy from the

antrum and corpus of the stomach were transported in thioglycolate broth to the microbiology

Chapter 6

96

laboratory. The specimens were rubbed on Belo Horizonte Medium (BHM) and on Campylobacter

selective medium (Becton Dickinson, Sparks, USA). Subsequently, the plates were incubated

microaerobically (Campypak, BBL, Becton Dickinson, Sparks, USA) at 35°C and were examined

three, five and seven days after incubation. Growth of H. pylori colonies was confirmed by typical

Gram stain, oxidase and urease activity. From all positive cultures, H. pylori colonies were

suspended in saline (3 McFarland) and plated on two seperate BHM agar plates. Each plate was

incubated with an E-test (AB-Biodisk, Solna, Sweden), one with clarithromycin and the other with

metronidazole. E-tests were read after two or three days microaerobical incubation (Campypak,

BBL, Becton Dickinson, Sparks, USA) at 35°C. Strains were considered resistant to metronidazole

if the minimal inhibitory concentration (MIC) was above 8 µl per ml. Resistance to clarithromycin

was considered to be present if the MIC of the strain was above 2 µl per ml.

The results of all H. pylori cultures and susceptibility tests performed from January 1st 1996 to

December 31st 2001 were collected. Subsequently, the prevalences of metronidazole and

clarithromycin resistance were calculated for each year and studied for any trends. To calculate the

expected efficacy of a 7-day PAM or PAC regimen, we used data from two meta-analyses [14,15].

Data on the total number of prescriptions for metronidazole and macrolides / lincosamides

(including clarithromycin) were collected from the Dutch “College voor Zorgverzekeringen”

covering about 65% of all Dutch inhabitants [21,22].

Secular trends in prevalence of both metronidazole and clarithromycin resistance were tested

for significance in Poisson regression models with resistance rates as dependent and time in years

as independent variable. A p value < 0.05 was considered significant.

Results

During the study period in total 16661 H. pylori cultures were performed. Seven (0.0008%)

cultures failed because of bacterial or fungal overgrowth and in six cases the results were lost,

leaving 16648 culture results for evaluation (99.9%). The presence of H. pylori was demonstrated

in 6231 (37.4%) cultures. The percentage of H. pylori cultures yielding a positive result remained

stable for each year over the study period (p=0.245). The results of susceptibility testing for

metronidazole and clarithromycin, and the data on total number of prescriptions for these drugs are

summarised in table 1.


97

Table 1.

The number of H. pylori positive cultures, and the prevalence of metronidazole and

clarithromycin resistance together with the number of prescriptions for these antibiotics in

the Netherlands.

Year 1996 1997 1998 1999 2000 2001

Total number of H. pylori cultures 2651 3072 3110 3248 2621 1959

Number of H. pylori positive cultures 1044 1094 1229 1287 915 662

Prevalence of metronidazole resistance (%) 28.4 27.1 19.3 14.1 11.3 13.5

Prevalence of carithromycin resistance (%) 2.2 1.7 1.2 3.3 2.8 2.1

• Total number of metronidazole

prescriptions (x1000)

10.48 9.88 9.21 8.61 8.58 8.7

• Total number of macrolide/lincosamide

prescriptions (x1000)

51 58 61 56 54 59

The prevalence of metronidazole resistance decreased from 28.4% in 1996 to 13.5% in 2001

and statistical analysis showed that this decreasing trend was significant over the study period

(p=0.011). The total number of prescriptions for metronidazole decreased significantly from

10.480 to 8700 during the study period (Pearson correlation coefficient = -0.914). The prevalence

of clarithromycin resistance fluctuated between 1.2% and 3.3% (mean 2.2%) without any

significant trend during the study period (p=0.49). The number of prescriptions for clarithromycin

remained about the same during the study period (Pearson correlation coefficient = 0.340).

Based on data from the meta-analysis by van der Wouden et al, the theoretical efficacy of the

PAM regimen in 100 consecutive H. pylori positive patients treated blindly with the PAM regimen

can be calculated (figure 1) [15]. The meta-analysis found that the efficacy of the PAM regimen

was 93% in patients harbouring a metronidazole susceptible strain and was lowered to 56% in case

a metronidazole resistant strain was present. When 100 consecutiveH. pylori positive patients have

to be treated in a region with a prevalence of metronidazole resistance of 13.5% (as in 2001), it can

be expected that 86.5 patients will carry a metronidazole susceptible strain. In these 86.5 patients,

the PAM regimen will have an efficacy of 93% resulting in 80.4 cured patients. In the

remaining13.5 patients, the PAM regimen will be less effective but will still result in 7.6 cured

Chapter 6

98

Figure 1.

The calculated efficacy of the protonpump inhibitor-amoxicillin-metronidazole (PAM)

regimen in 100 consecutive patients treated blindly for H. pylori at a prevalence of

metronidazole resistance of 13.5%.

100 H. pylori positive patients

Infected with metronidazole

susceptible strain

Infected with metronidazole resistant

strain

Efficacy of PAM in case of

metronidazole susceptible strain

Efficacy of PAM in case of

metronidazole resistant strain

Number of eventually cured patients

80.4 patients

Number of eventually cured patients

7.6 patients

Total cured patients (cure rate %)

88 patients (88%)


99

patients. Therefore, the overall efficacy of the PAM regimen will be 88.0%. Based on this model,

the influence of changes in the prevalence of metronidazole resistance on the overall cure rate of

the PAM regimen can be calculated and is shown in figure 2.

The same calculation can be applied to the PAC regimen using the data from the meta-analysis

by Houben et al. The calculated overall efficacy of the PAC regimen at a clarithromycin resistance

rate of 2.1% (as in 2001) is 80.3%. Figure 3 shows how changes in the prevalence of

clarithromycin resistance affect the overall efficacy of the PAC regimen.

Figure 2.

The influence of changes in the prevalence of metronidazole resistance on the overall efficacy

of the PAM regimen prescribed in patients with unknown Helicobacter pylori susceptibility

status. (The cross marks the current situation)

Eff

icac

y of

the

PAM

regi

men

→

Prevalence of metronidazole resistance →

50%

60%

70%

80%

90%

100%

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

x

Chapter 6

100

Figure 3.

The influence of changes in the prevalence of clarithromycin resistance on the overall

efficacy of the PAC regimen prescribed in patients with unknown Helicobacter pylori

susceptibility status. (The cross marks the current situation)

Eff

icac

y of

the

PAC

regi

men

→

Discussion

This study showed that in the northeastern part of the Netherlands the prevalence of

metronidazole resistance in H. pylori decreased significantly from 1996 to 2001, while the

prevalence of clarithromycin resistance remained stable.

The observed decrease in the number of prescriptions for metronidzole may explain the

decrease in the prevalence of metronidazole resistance. It is to be noted that most prescriptions for

metronidazole are no part of an anti-H. pylori regimen, but are used for other conditions like

gynaecological infections and abdominal surgery [23,24]. In a previous publication by our group, a

rapid increase in the prevalence of metronidazole resistance was reported during the period 1993

to 1996 [25]. We have no consistent explanation for these discrepant findings, but it is striking that

during that period a slight increase in the number of metronidazole prescriptions was observed. It

is evident that the decreasing trend in metronidazole resistance will increase the efficacy of the

Prevalence of clarithromycin resistance →

75%

76%

77%

78%

79%

80%

81%

82%

83%

84%

85%

0% 1% 2% 3% 4% 5% 6% 7% 8% 9% 10%

x


101

PAM regimen in the future. Even at the time of the highest prevalence of metronidazole resistance

in 1996, however, the PAM regimen still had an efficacy of 82.5%, meeting the recommendations

of the European Society of Primary Care Gastroenterology.

The prevalence of clarithromycin resistance remained stable between 1996 to 2001 and is still

quite low as compared to other European countries [26-28]. Data from the “College voor

Zorgverzekeraars” also did not show any changes in the yearly number of prescriptions for

macrolides and lincosamides during the study period. It has to be kept in mind, however, that the

low prevalence of clarithromycin resistance in this part of the Netherlands may prohibit the

detection of trends (type II error). Despite the low prevalence of clarithromycin resistance, the

calculated efficacy of the PAC regimen was only 80.3% and thereby only just above the cut-off

value of 80% as recommended by the European Society of Primary Care Gastroenterology. As a

result of the great impact of clarithromycin resistance on the efficacy of clarithromycin containing

anti-H. pylori regimens, even a small increase in the prevalence of clarithromycin resistance (to >

3.0%) will result in a drop in expected efficacy of the PAC regimen to 79.9%. Remarkably, the

European Society of Primary Care Gastroenterology has recommended the PAC regimen as the

first choice for blind anti-H. pylori therapy [13]. Based on our calculations, however, it is clear

that the PAM regimen currently has a higher efficacy than the PAC regimen, despite the much

higher prevalence of metronidazole resistance as compared to clarithromycin resistance.

Several other studies have described changes in the prevalence of metronidazole and/or

clarithromycin resistance in H. pylori [27]. Most of these studies found that the prevalence of

metronidazole and clarithromycin resistance increased during the past decade [17-19,27,29-32].

Although it has often been suggested that this increase is caused by an increased use of antibiotics,

only two studies have actually correlated the use of these antibiotics to the prevalence of

antimicrobial resistance for these drugs. In one study an increase in the prevalence of H. pylori

resistance to macrolides was not associated with an increased use of these drugs [20]. Another

study concluded that the increase in both metronidazole and clarithromycin resistance in H. pylori

was associated with an increased use of these antibiotics [32]. To our knowledge, the data in our

study represent the most recent data on prevalences of metronidazole and clarithromycin resistance

in H. pylori (up to 2001). Whether the observed decrease in the prevalence of metronidazole

resistance is really related to the decreased use of metronidazole in our region, or is part of a

natural fluctuation remains to be seen.

Chapter 6

102

In conclusion, over the period 1996 to 2001, the prevalence of metronidazole resistance

decreased significantly in the northeastern part of the Netherlands, whereas the prevalence of

clarithromycin resistance remained stable. The decrease in the prevalence of metronidazole

resistance may be related to a decrease in the total number of metronidazole. Currently, it is still

possible to achieve a cure rate of at least 80% with both the PAM and PAC regimen when treating

H. pylori infections blindly in primary care. When the decreasing trend in metronidazole resistance

is consistent it will increase the efficacy of the PAM regimen in the future. On the other hand,

even a slight increase (<1%) in the prevalence of clarithromycin resistance will reduce the efficacy

of the PAC regimen to less than 80%. To ensure that blind treatment of H. pylori infections with a

protonpump inhibitor based triple regimen remains effective in the future, continuous monitoring

the prevalence of metronidazole and clarithromycin resistance is necessary.

References

1. EUROGAST study group. Epidemiology of, and risk factors for, Helicobacter pylori infection among

3194 asymptomatic subjects in 17 populations. Gut 1993; 34: 1672-1676.

2. Marshall BJ. Helicobacter pylori. American Journal of Gastroenterology 1994; 89: S116-S127.

3. EUROGAST study group. An international association between Helicobacter pylori infection and

gastric cancer. Lancet 1993; 341: 1359-1362.

4. Uemura N, Okamoto S, Yamamoto S, Matsumura N, Yamaguchi S, Yamakido M et al.

Helicobacter pylori infection and the development of gastric cancer. New England Journal of Medicine

2001; 345: 784-789.

5. Blaser MJ. Not all Helicobacter pylori strains are created equal: should all be eliminated ? Lancet 1997;

349: 1020-1022.



2000; 356: 455-460.



8. Price AB. Histological aspects of Campylobacter pylori colonisation and infection of gastric and

duodenal mucosa. Scandinavian Journal of Gastroenterology 1988; 142: 21-24.

9. Lam SK, Talley NJ. Report of the 1997 Asia pacific consensus conference on the management of

Helicobacter pylori infection. Journal of Gastroenterology and Hepatology 1997; 13: 1-12.


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10. Talley NJ, Axon A, Bytzer P, Holtmann G, Lam SK, Zanten SV. Management of uninvestigated and

functional dyspepsia: a working party report for the World Congress of Gastroenterology 1998.

Alimentary Pharmacology & Therapeutics 1999; 1135-1148.

11. Delaney BC, Innes MA, Deeks J, Wilson S, Cooner MK, Moayyedi P et al. Initial management

strategies for dyspepsia. Cochrane Database Syst Rev 2001; 3: CD001961.

12. Malfertheiner P, Megraud F , O'Morain C, Hungin APS, Jones R, Axon A et al. Current concepts in

the management of Helicobacter pylori infection. The Maastricht 2-2000 consensus report. Alimentary

Pharmacology & Therapeutics 2002; 16: 167-180.

13. European Society of Primary Care Gastroenterology. The management of Helicobacter pylori

infection. Consensus meeting, 9-10 may, Zurich 1998.

14. Houben MH, Van Der BD, Hensen EF, Craen AJ, Rauws EA, Tytgat GN . A systematic review of

Helicobacter pylori eradication therapy. The impact of antimicrobial resistance on eradication rates.


15. van der Wouden EJ, Thijs JC, van Zwet AA, Sluiter WJ, Kleibeuker JH. The influence of in vitro

nitroimidazole resistance on the efficacy of nitromidazole containing anti-Helicobacter pylori regimens:

a meta-analysis. American Journal of Gastroenterology 1999; 94: 1751-1759.

16. van Zwet A, Vandenbroucke-Grauls CMJE, Thijs J, van der Wouden EJ, Gerrits MM, Kusters

JG. Stable amoxicillin resistance in Helicobacter pylori. Lancet 1998; 352: 1595.

17. Lang TKW, Cheng AFB, Sung JJY, Yui PYL, Chung SSC. An increase in Helicobacter pylori

strains resistant to metronidazole: A five-year study. Helicobacter 1996; 1: 57-61.

18. de Koster E, Devaster JM, Van den Borre C, Delaunoit T, Langlet P, Deltrene M. A nine year

surveillance of Hp resistance to macrolides and imidazoles. Gastroenterology 1999; 116: A145.

19. Lopez-Brea M, Martinez MJ, Domingo D, Alarcon T. A 9 year study of clarithromycin and

metronidazole resistance in Helicobacter pylori from Spanish children. Journal of Antimicrobial

Chemotherapy 2001; 48: 295-297.

20. Bontems P, Devaster JM, Corvaglia L, Dezsofi A, Van den BC, Goutier S et al. Twelve year

observation of primary and secondary antibiotic-resistant Helicobacter pylori strains in children.

Pediatric Infectious Diseases Journal 2001; 20: 1033-1038.

21. College voor Zorgverzekeringen . Genees- en hulpmiddelen Informatie Project (GIP). GIP-peilingen

1996 t/m 2001 2002; 10, 12, 14, 16, 18, 20.

22. College voorZorgverzekeringen . Genees- en hulpmiddelen Informatie Project (GIP). GIP-peilingen,

metronidazol voorschriften 1996 t/m 2002 2002.

23. Rautelin H, Seppala K, Renkonen OV, Vainio U, Kosunen TU. Role of metronidazole resistance in

therapy of Helicobacter pylori infections. Antimicrobial Agents and Chemotherapy 1992; 36: 163-166.

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24. Glupczynski Y. Results of a multicentre European survey in 1991 of metronidazole resistance in

Helicobacter pylori. European Journal of Clinical Microbiology & Infectious Diseases 1992; 11: 777-

781.

25. van der Wouden EJ, van Zwet AA, Vosmaer GD, Oom JA, de Jong A, Kleibeuker JH. Rapid

increase in the prevalence of metronidazole-resistant Helicobacter pylori in the Netherlands. Emerging

Infectious Diseases 1997; 3: 385-9.

26. Megraud F. Resistance of Helicobacter pylori to antibiotics. Alimentary Pharmacology & Therapeutics

1997; 11: 43-53.

27. Lopez-Brea M, Domingo D, Sanchez I, Alarcon T. Evolution of resistance to metronidazole and

clarithromycin in Helicobacter pylori clinical isolates from Spain. Journal of Antimicrobial

Chemotherapy 1997; 40: 279-81.

28. Glupczvnski Y, Megraud F, Andersen LP, Lopez-Brea M. Antibiotic susceptibility of H.pylori in

Europe in 1998: results of the third multicentre study. Gut 1999 ; 45 (suppl 3): A3.

29. Glupczynski Y, Goutier S, van de Borre C, Butzler JP, Burette A. Surveillance of Helicobacter

pylori resistance to antimicrobial agents in Belgium from 1984 to 1994. Gut 1995; 37 (suppl 1): A97.

30. Weissfeld AS, Simmons DE, Vance PH, Trevino E, Kidd S, Greski-Rose P. In vitro susceptibility of

pre-treatment isolates of Helicobacter pylori from two multicenter United States clinical trials.


31. Perez AL, Kato M, Nakagawa S, Kawarasaki M, Nagasako T, Mizushima T et al. The relationship

between consumption of antimicrobial agents and the prevalence of primary Helicobacter pylori

resistance. Helicobacter 2002; 7: 306-309.

32. Cabrita J, Oleastro M, Matos R, Manhente A, Cabral J, Barros R et al. Features and trends in

Helicobacter pylori antibiotic resistance in Lisbon area, Portugal (1990-1999). Journal of Antimicrobial

Chemotherapy 2000; 46: 1029-1031.

105

Chapter 7

The importance of vacA , cagA, and iceA genotypes of Helicobacter

pylori infection in peptic ulcer disease and gastroesophageal reflux

disease.

Nicolaas L. A. Arents 1, Anton A. van Zwet 1, Jacob C. Thijs 2, Anne M.D. Kooistra-

Smid 1, Kornelis R. van Slochteren 1, John E. Degener 3, Jan H. Kleibeuker 1, and Leen-

Jan van Doorn 5.

1 Regional Public Health Laboratory, Groningen / Drenthe2 Department of Internal Medicine, Bethesda Hospital, Hoogeveen3 Department of Medical Microbiology, University Hospital, Groningen4 Department of Gastroenterology, University Hospital, Groningen5 Delft Diagnostic Laboratory, Delft

Published in American Journal of Gastroenterology 2001; 96: 2603-2608.

Chapter 7

106

Abstract

Objective

To study the relationship between the presence of H. pylori virulence factors and clinical outcome

in H. pylori infected patients.

Methods

DNA was isolated from an antral biopsy sample and vacA, cagA, and iceA genotype were

determined by PCR and a reverse hybridization technique in 183 patients with culture-proven H.

pylori infection: 51 with peptic ulcer disease (PUD), 62 with gastroesophageal reflux disease

(GERD), and 70 with a normal endoscopy (gastritis only; GO).

Results

Forty-four samples (24 %) showed more than one allelic variant in the vacA s- or m-region and/or

both iceA1 and iceA2 genotypes, indicating multiple strain infection. These were excluded from

statistical analysis. vacA s1 and cagA were significantly more common in PUD than in GERD and

GO. Logistic regression analysis showed that GERD patients were more often infected with strains

lacking both cagA and iceA than GO patients (OR = 0.36; CI = 0.15 - 0.89). Trend analysis

showed that GERD patients were most often infected with less virulent strains (p < 0.002).

Conclusions

Multiple strain infection is common. H. pylori strains possessing the vacA s1 genotype and/or

cagA are associated with PUD. GERD patients, infected with H. pylori mostly carry less virulent

strains possessing neither cagA nor iceA1. Our findings support the hypothesis that virulent strains

protect against the development of GERD.

The importance of the vacA, cagA, and iceA genotype

107

Introduction

Helicobacter pylori (H. pylori) infection is highly prevalent, especially in the developing world

where the large majority of the population is infected. In the western world the infection rate is

gradually decreasing but prevalences still range from 25 to 50% [1]. The majority of the infected

population, however, never develops clinically significant disease [2]. Only about 10 % of the

infected subjects will eventually develop peptic ulcer disease (PUD) and 1-2 % a gastric

malignancy [3,4].

The reason for the variable outcome of the infection is still unclear, but it is likely that three

elements are involved. First, host factors like the HLA system and receptors to Lewis antigens

have been suggested to influence clinical outcome [5]. Second, environmental factors, such as

smoking and food, may be important [6-8]. Third, as with other gastrointestinal pathogens [9,10],

the clinical outcome probably depends on differences in the virulence of the infecting strain. In

this study, the importance of several putative virulence factors in the development of H. pylori

related disease, was evaluated.

Several H. pylori genes associated with pathogenicity have been identified [11]. The vacA

gene, encoding a vacuolating cytotoxin (VacA) is present in all strains, but only about 50 % of

these strains produce a detectable active toxin [12]. This depends on the type of sequences within

the signal and middle region (s- and m-region) of the vacA gene [13]. The s-region contains one of

the following allelic subtypes: s1a, s1b, s1c or s2. The m-region contains either the m1 or the m2

allele. Previous studies have shown that vacA s1/m1 strains produce large amounts of vacuolating

cytotoxin and that these strains are associated with PUD [13-17]. On the other hand, the s2/m2

strains produce no or only small amounts of cytotoxin and are uncommon in patients with PUD

[13]. The s1/m2 strains seem to take an intermediate position.

The cytotoxin associated gene (cagA) is responsible for the production of the CagA protein, the

function of which is not yet understood. However, cagA is considered as a marker for the presence

of a genomic pathogenicity island [18]. This island contains other genes enhancing the virulence of

the infecting strain, such as picB, known to promote the induction of cytokines by the host [19,20].

Several studies have shown that cagA positive H. pylori strains cause more extensive epithelial

damage [21,22]. Other studies have indicated an association between the presence of cagA and

PUD [17,23-27].

Chapter 7

108

Recently, another virulence-associated gene was described [28]. This designated iceA gene

("induced by contact with epithelium") has two allelic variants (iceA1 and iceA2). The iceA1

variant is upregulated upon contact of the bacterium with epithelial cells and seems to be

associated with PUD [17,28].

In this study the relationship between these three virulence-associated factors and clinical

outcome was investigated. As recent studies have suggested that infection with a virulent (cagA-

positive) H. pylori strain may in fact protect against the development of gastroesophageal reflux

disease (GERD), we also included biopsy samples obtained from patients with GERD [29-31].

Furthermore, as infection with more than one strain is rather common [32-35], and false

conclusions can be drawn if only a few colonies of a culture are examined [36,37], we used whole

biopsy specimens, to be sure that all H. pylori DNA was included.

Materials and methods

In all patients undergoing upper gastrointestinal endoscopy for dyspepsia in a Dutch regional

hospital, antral biopsy specimens were taken for H. pylori culture as described previously [38]. All

culture-positive biopsy samples were frozen and stored at -70o C. From this collection, consecutive

antral biopsy specimens were selected to form three predefined groups: samples from patients with

peptic ulcer disease (PUD), samples from patients with gastroesophageal reflux disease (GERD),

and samples from patients with gastritis only or just an hiatal hernia (gastritis only; GO). The PUD

group included patients with an active ulcer (mucosal break > 5 mm with clearly visible ulcer

crater) or evident deformation of the duodenal bulb at endoscopy as well as patients with a history

of endoscopically or radiologically documented ulcer disease. Patients with endoscopic signs of

GERD (visible erosions or Barrett's metaplasia) and patients using non-steroidal anti-inflammatory

drugs (NSAIDs) were excluded from the PUD group. The GERD group included patients with

visible erosions at endoscopy (Savary-Miller grade 1 or higher) or Barrett's metaplasia. Patients

with a PUD history or endoscopic signs of PUD were excluded from this group. Patients with

previous H.pylori eradication treatment, the use of H2-receptor antagonists in the two weeks

before endoscopy, or the use of proton pump inhibitors in the four weeks preceding endoscopy

were discarded in all groups. None of these patients participated in any previous study regarding

virulence factors.


109

Extraction and isolation of DNA was performed on each entire biopsy specimen according to

Boom et al. [39] or by the phenol-chloroform extraction method [40]. For the detection of cagA

and the determination of the vacA and iceA genotype, isolated DNA was amplified by PCR in a

Thermocycler (MJ Research, PTC 200) as described [35]. The PCR conditions were: 9 min pre-

incubation at 94ºC, followed by 40 cycles of 30 sec at 94ºC (denaturation), 45 at 50ºC (annealing)

and 45 at 72ºC (extension). A final extension was performed for 5 min at 72ºC [17].

To detect the presence of cagA and to determine the vacA s- and m-genotypes, a single step

reverse hybridization technique ( prototype line probe assay, LiPA, Innogenetics NV, Gent,

Belgium) was used. This assay comprises a nitrocellulose strip containing multiple specific

oligonucleotide probes for the vacA s-region (s1a, s1b, s1c and s2), the vacA m-region (m1 and

m2) and the cagA gene [35], enabling the simultaneous detection of all these alleles. For each of

the genotypes, two specific probes were present on the strip, permitting accurate genotyping. The

iceA genotype was determined by type-specific PCR and standard agarose gel electrophoresis. The

iceA primers generated a 210 bp amplificate for the iceA1 genotype and a 124, 229, 334 or 439 bp

amplificate for the iceA2 genotype.

If more than one allele of either the s-region or the m-region of vacA was detected or if biopsy

specimens showed both the iceA1 and iceA2 genotype, it was concluded that the biopsy specimen

harbored at least two different strains. These specimens were excluded when studying the

relationship between virulence factors and clinical outcome. To compare differences between two

groups, statistical analysis was performed using the Chi-square test with Yates' correction. Trends

were analyzed by the Mann-Whitney U-test. Furthermore, logistic regression analysis was

performed to evaluate which virulence factors contributed significantly in predicting the

underlying disease (Sysstat 7.0). Results are presented as odds ratios (OR) with 95% confidence

intervals (CI). For the Mann-Whitney U-test, a p value < 0.05 was considered significant.

Chapter 7

110

Results

One hundred and eighty three biopsy specimens, all obtained from different patients were

studied. The PUD group included 51 specimens, the GERD group of 62 specimens, and the GO

group of 70 specimens. The age of the patients was similar in all groups. PUD patients were more

frequently male than GO patients (OR = 2.38; CI = 1.07 - 5.35). Severity of the esophagitis in the

GERD group, according to the Savary-Miller classification, was grade 1 in 50, grade 2 in six,

grade 3 in two and grade 4 in three patients. Barrett's dysplasia was encountered in 7 patients, 1 of

whom had no active esophagitis (grade 0).

Based on the vacA and iceA genotypes, 138 specimens (75%) were colonized by a single H.

pylori strain. This group includes five biopsy specimens that did not show any iceA genotype. One

biopsy specimen did not yield a result for the m-region and could therefore not be classified. The

remaining 44 biopsy specimens (24%) harbored more than one strain. In 26 (14%) of these cases,

multiple vacA genotypes were detected, but only a single iceA genotype was present. Conversely,

ten biopsy specimens (5%) showed both iceA genotypes, but only a single genotype for the vacA

region. Eight samples (6%) revealed multiple alleles for both vacA and iceA. The prevalence of

multiple strain infection was not significantly different between the three groups (PUD: 22%; GO:

29%; GERD: 21%, n.s.).

Table 1 shows all encountered vacA genotype combinations per group after exclusion of biopsy

specimens containing multiple strains. In accordance with the findings of others [13], we did not

detect the s2/m1 genotype. The vacA s1 genotype (vacA s1a, s1b or s1c) was present in 58% of all

biopsy specimens and was significantly more common in PUD than in GO and GERD (PUD:

88%; GO: 51%; GERD: 41%; OR = 6.72; CI = 2.08 - 25.14 and OR = 10.15; CI = 3.12 - 37.95

respectively). The vacA s1 genotype seemed less prevalent in GERD than in GO, but this

difference was not significant (OR = 0.66; CI = 0.28 - 1.58). The vacA m1 genotype was present in

34 (25%) of the 138 biopsy specimens. The prevalence of the vacA m1 genotype did not differ

between the three groups (PUD: 33%; GO: 29%; GERD: 14%; n.s.).

The cagA gene was present in 64 (46%) of the 138 biopsy specimens and was most common in

PUD (70%) and less common in GO (43%) and GERD (31%). The prevalence of cagA was higher

in PUD than in GO and GERD, (OR = 3.11; CI = 1.19 - 8.31 and OR = 5.29; CI 1.95 - 14.56

respectively). The cagA genotype seemed less prevalent in GERD than in GO, but statistical

analysis did not show a difference (OR = 0.59; CI = 0.24 - 1.46).


111

Table 1.

Prevalence of different vacA genotypes in the three groups for single strain harboring biopsy

specimens (n=138).

vacA

genotype

s1a-

m1

s1a-

m2

s1b-

m1

s1b-

m2

s1c-

m1

s1c-

m2

s2-

m2

total

PUD 11 20 1 1 1 1 5 40

GO 13 9 1 2 0 0 24 49

GERD 7 13 0 0 0 0 29 49

total 31 42 2 3 1 1 58 138

PUD = peptic ulcer disesae, GO = gastritis only, GERD = gastroesophageal reflux disease

Table 2 shows the distribution of the iceA genotypes in single strain harboring biopsy

specimens. Five samples (4%) did not show either iceA1 or iceA2, and were omitted when the

relationship between the iceA genotype and clinical outcome was studied. The iceA1 genotype was

present in 58 (44%) of the remaining 133 biopsy specimens (PUD: 60 %; GO: 47 %; GERD: 29

%) and was significantly more prevalent in PUD than in GERD (OR = 3.75; CI = 1.37 - 10.40).

The differences between PUD and GO and between GO and GERD, however, were not significant

(OR = 1.70; CI = 0.65 - 4.49 and OR = 0.45; CI = 0.18 - 1.13 respectively). The prevalences of the

various virulence factors in the three groups are shown in Figure 1.

Table 2.

iceA genotypes present in the three groups for single strain harboring biopsy specimens

(n=138).

iceA genotype A1 A2 none total

PUD 21 14 5 40

GO 23 26 0 49

GERD 14 35 0 49

total 58 75 5 138

PUD = peptic ulcer disesae, GO = gastritis only, GERD = gastroesophageal reflux disease

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112

Figure 1.

Prevalence of vacA s1, cagA and iceA1 genotype in peptic ulcer disease (PUD; n = 40),

gastritis only (GO; n = 49) and gastroesophageal reflux disease (GERD; n = 49). Only biopsy

specimens harboring one strain are included.

Figure 2.

Prevalence of different combinations of cagA and iceA1 in 49 H. pylori positive patients with

GERD. The trend is significant (p < 0.002).

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

PUD GO GERD

vaca s1 cagA+ iceA1

0%

10%

20%

30%

40%

50%

60%

cagA+ / iceA 1 cagA+ or iceA 1 cagA- / iceA 2


113

To investigate what virulence factor was most predictive for underlying disease, logistic

regression analysis was performed. It showed that vacA s1 was the only factor predictive for PUD

(OR = 3.0; CI 1.73 - 5.28), no factor was predictive for GO, and both cagA and iceA1 were

negatively associated with GERD (OR = 0.44; CI = 0.21 - 0.91 and OR = 0.42; CI = 0.19 - 0.90

respectively). When all virulence factors were considered separately, no differences were found

between GO and GERD. When, however, both cagA and iceA1 were taken into account, GERD

patients proved to be significantly more often infected with strains showing none of these factors

(OR = 0.36; CI = 0.15 - 0.89); vacA genotype was left out from this analysis as logistic regression

did not show a predictive value for this factor in GO and GERD).

To test the hypothesis that patients with GERD are usually infected with less virulent strains,

we examined how often these patients were infected with strains possessing both cagA and iceA1,

either cagA or iceA1, or neither of these factors (figure 2). The Mann-Whitney U-test showed a

significant trend: GERD patients were most often infected with strains possessing none of these

factors and strains showing both factors were least common (p < 0.002).

Discussion

The present study investigated the relationships between various virulence factors (vacA, cagA

and iceA) of H. pylori and the clinical outcome of the infection, using the line probe assay (LiPA)

that enables the detection of multiple PCR-amplified products at the same time.

As the vacA gene contains only one allele for the s-region and one for the m-region in each H.

pylori strain [13], the presence of more than one line for either region on the LiPA strip suggests

that the specimen harbors more than one strain. Moreover, since each H. pylori strain only

possesses a single iceA locus the detection of both iceA1 and iceA2 in a single biopsy specimen

also indicates the presence of multiple strains.

Our study revealed a high prevalence of multiple strain infection (24 %). In a previous study,

using the same method, multiple strain infection was found in 29 % of a Portuguese and in 8 % of

a Dutch population [35]. However, other studies using various methods for the detection of

multiple strain infection, found this phenomenon in only 7 - 13 % [32,33,41] of samples. These

discrepancies are likely to be due to sampling error, but geographic differences can also be

responsible. While in some studies, like ours, the entire biopsy specimen was used, in others only a

few H. pylori colonies were examined [32,33,41]. It is possible, however, that even in our study

Chapter 7

114

the prevalence is an underestimation, since the detection of multiple strains was solely based on

the presence of multiple vacA and iceA genotypes. Moreover, we only examined one biopsy

sample. Although it is still debated whether or not the bacterial population in one biopsy sample is

representative for the entire stomach population, some suggest that strain-specific colonisation of

different regions within the stomach occurs [33,36,37,42-45]. Considering the frequent occurrence

of multiple strain infection it can be easily imagined that false conclusions will be drawn on the

associations between virulence factors and clinical outcome if only a small number of bacterial

colonies, not representative for the entire stomach population, are examined [36,37]. As mentioned

above, we therefore used the whole biopsy specimen for DNA isolation and excluded samples

shown to harbor more than one H. pylori strain.

Our study suggests that both vacA s1 and cagA are associated with PUD, as these factors were

significantly more common in the PUD group than in both other groups. In that respect our data

are in agreement with the findings of several other investigators and suggest a possible role of

these factors in the pathogenesis of H pylori related peptic ulcer disease [17,23-27]. In predicting

underlying disease, a logistic regression analysis, however, only pointed at vacA s1 as a predictive

factor. This suggests that the presence of cagA is not independently associated with PUD, but

associated with vacA s1. Our results do not point at vacA m1 as a significant virulence factor.

iceA1 seemed to be more common in PUD than in both other groups, only the difference with

GERD was statistically significant.

Recently, it has been suggested that H. pylori infection may be protective against GERD

[30,31,46,47] and that H. pylori positive GERD patients (especially those with the more serious

manifestations of the disease) are infected with less virulent strains [29]. Our data seem to support

this view. All putative virulence factors seemed to be more common in GO than in GERD

although the differences never reached statistical significance. As the groups were relatively small,

however, a type II error can not be excluded. Moreover, when we took the two factors, shown by

logistic regression to be markers for the absence of GERD, both into account (cagA and iceA1),

GERD patients proved to be significantly more often infected with strains showing neither of these

factors than patient with GO. Finally, the Mann-Whitney U-test showed that there was a

significant trend: our H.pylori positive GERD patients were most often infected with strains

showing neither of these two factors and least often with strains possessing both factors.


115

In conclusion, our study performed in a rural region of The Netherlands, shows that multiple

strain infection is rather common (24%). PUD is associated with the presence of H. pylori strains

of the vacA s1 genotype that in most cases are also cagA-positive. On the other hand, GERD

patients, infected with H. pylori mostly carry less virulent strains possessing neither cagA nor

iceA1. Our findings support the hypothesis that virulent strains protect against the development of

GERD.

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Chapter 8

The value of PCR and serology based methods in determining the

presence of the cagA gene in Helicobacter pylori infection.

Nicolaas L. A. Arents 1, Anton A. van Zwet 1, Jacob C. Thijs 2, Raymond G. Pot 3,

Jan H. Kleibeuker 4, John E. Degener 5, and Johannes G. Kusters 3.

1 Regional Public Health Laboratory, Groningen / Drenthe2 Department of Internal Medicine, Bethesda Hospital, Hoogeveen3 Department of Gastroenterology, Erasmus MC University Medical Center, Rotterdam4 Department of Gastroenterology, University Hospital, Groningen5 Department of Medical Microbiology, University Hospital, Groningen


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Abstract

Background

To compare PCR, OroVax CagA protein recombinant based ELISA, and a recently introduced

rapid serological test for anti-CagA antibodies (CagA rapid test, Orion Diagnostica) for the

detection of the cagA gene of Helicobacter pylori.

Materials and Methods

PCR followed by hybridisation was compared to the frequently used OroVax CagA recombinant

based ELISA in 91 culture proven H. pylori positive patients and 50 culture and histology proven

H. pylori negative controls. The CagA rapid test (Orion) was compared to a surrogate gold

standard based on the samples of patients showing concordant PCR and OroVax CagA

recombinant based ELISA results.

Results

PCR with hybridisation and OroVax CagA recombinant based ELISA showed concordant results

in 118 patients. In the remaining patients, PCR was positive in 22 patients and OroVax CagA

recombinant based ELISA was positive in 1 patient. When the CagA rapid test (Orion) was

compared to the surrogate gold standard, the test showed very accurate results : sensitivity =

96.8%, specificity = 95.4%, and accuracy = 95.8%.

Conclusions

Although a reliable gold standard for the detection of cagA is lacking, the CagA rapid test (Orion)

provides a very accurate and easy to perform tool when compared to PCR with hybridisation and

OroVax CagA recombinant based ELISA.

The value of PCR and serology based methods in determining the presence of cagA

121

Introduction

Infection of the gastric mucosa with Helicobacter pylori (H. pylori) is highly prevalent and

occurs worldwide. The clinical consequences of this infection, however, are remarkably variable

and while most infected subjects are asymptomatic others suffer from significant disease, such as

peptic ulcer disease (PUD) or gastric cancer [1]. H. pylori DNA profiles indicate a considerable

amount of genomic variation between unrelated strains [2,3] and as a consequence, most patients

harbour unique strains [4,5]. This genetic diversity is likely to play an important role in the wide

range of clinical outcomes as several distinct alleles of H. pylori genes were shown to be

associated with the more severe disease types of the upper alimentary tract [6]. These genes, or so-

called virulence factors, include vacA, iceA, and cagA. While the vacA and iceA genes are present

in all strains, the cagA gene (cytotoxin associated gene), is present only in about one out of two H.

pylori strains [7]. The cagA encoded CagA protein is highly immunogenic in humans and thought

to induce an antibody response in virtually all infected subjects [8]. CagA is probably not a

virulence factor itself, but represents a convenient marker for the presence of the Cag

pathogenicity island [9-11] that contains other genes enhancing the virulence of the infecting strain

[12]. Although it is not a virulence factor itself, CagA has been the subject of many studies. The

reliability of these studies, however, depends on the accuracy of the methods used to detect the

presence of CagA and the latter is complicated because a firm gold standard is lacking.

In this study, we evaluated the agreement between PCR and a frequently used anti-CagA

antibody ELISA based on the orv220 antigen from OroVax (OroVax, Inc, Cambridge, MS, USA)

[13]. Based on these results, a reliable gold standard could be constructed and this was then used to

evaluate the accuracy of a recently introduced anti-CagA antibody rapid test (Orion Diagnostica,

Espoo, Finland).

Methods

Patients

The study was performed in a regional hospital in the northern part of The Netherlands. In all

patients undergoing upper gastrointestinal endoscopy for dyspepsia in this hospital, antral biopsy

samples are routinely taken for H. pylori culture and histology (HE and Giemsa stain). H. pylori

positive specimens are frozen and stored at -70oC. For 91 of these specimens, we also had a frozen

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122

serum sample (obtained immediately before endoscopy), and this set was used in this study. The

control group consisted of serum and biopsy samples of 50 individuals, that were shown to be H.

pylori negative by both culture and histology.

PCR detection of the cagA gene

The entire biopsy specimens (only from the H. pylori positive patients) were used to extract

and isolate H. pylori DNA according to Boom et al. [14]. The isolated DNA was amplified in a

Thermocycler (MJ Research, PTC 200). A PCR procedure amplified the cagA gene, if present,

using the following primers: TTGACCAACAACCACAAACCGAAG (F (+)) and

CTTCCCTTAATTGCGAGATTCC (R (-)) [15]. PCR conditions were: 9 minutes pre-incubation

at 94ºC, followed by 40 cycles of 30 seconds at 94ºC (denaturation), 45 at 50ºC (annealing) and 45

at 72ºC (extension). A final extension was performed for 5 minutes at 72ºC. To detect the presence

of cagA amplificate a single step reverse hybridisation technique (prototype line probe assay,

LiPA, Innogenetics NV, Gent, Belgium) was used. This assay comprises a nitro-cellulose strip

containing two different specific oligonucleotide probes for the cagA gene, permitting a

convenient hybridisation based confirmation of the cagA PCR product [16].

Serological testing for anti-H. pylori IgG antibodies and anti-CagA antibodies

All serum samples were tested for the presence of anti-H. pylori IgG antibodies using a

commercially available assay (Pyloriset EIA-G III, Orion Diagnostica, Espoo, Finland). The

presence of anti-CagA antibodies was determined by two different serological tests. An in-house

standard ELISA was performed based on the recombinant orv220 antigen from OroVax (OroVax,

Inc, Cambridge, MS, USA) [13] and results were expressed as optical densities (OD). We used the

cut-off value as determined in a previous study, based on the upper limit of two standard

deviations from the mean in a large H. pylori negative Dutch population (OD = 0.458 nm) [17].

The CagA rapid test (Orion) is an immunochromatographic test acquiring a single drop of serum

or whole blood to show a positive or negative result within 10 minutes. Sensitivity, specificity, and

accuracy were calculated in the usual way. As positive and negative predictive value have no

meaning in this selected population, these were omitted. The CagA rapid test (Orion) was

compared to the serum samples in which PCR and the OroVax CagA recombinant based ELISA


123

were in agreement (gold standard). Statistical comparison was performed by Chi-square testing

using Yates correction and p < 0.05 was considered significant.

Results

Comparison of PCR and OroVax CagA recombinant based ELISA

PCR detected cagA positive H. pylori strains in 53 (58%) of the 91 H. pylori positive biopsy

specimens (table 1). The OroVax CagA recombinant based ELISA detected anti-CagA antibodies

in 31 (34.1%) of the 91 H. pylori positive biopsy specimens and was negative in 49 (98.0%) of the

50 H. pylori negative biopsy specimen. PCR and the OroVax CagA recombinant based ELISA

were in agreement in 118 (83.7%) of the 141 patients (table 1). IgG anti-H. pylori antibodies were

detected in 78 (85.7%) of the 91 H. pylori positive patients. The 13 false negative IgG results

occurred in eight patients with a negative result for both PCR cagA and OroVax CagA

recombinant based ELISA, in four patients with only a positive PCR cagA, and in one patients

with both positive PCR cagA and OroVax CagA recombinant based ELISA. The IgG anti-H.

pylori antibody test was negative in 47 (94.0%) of the 50 H. pylori negative patients. In one of the

three samples with a false positive result, the OroVax CagA recombinant based ELISA was also

false positive.

Evaluation of the CagA rapid test (Orion)

The accuracy of the CagA rapid test (Orion) was evaluated by constructing a gold standard

based on the serum samples and biopsy specimens in which PCR and the OroVax CagA

recombinant based ELISA were in agreement. The CagA rapid test (Orion) identified 30 (96.8%)

of the 31 cagA positive and 83 (95.4%) of the 87 cagA negative serum samples. The test was

negative in 48 (96.0%) of the 50 H. pylori negative patients. Changing the cut-off value for the

CagA rapid test (Orion) did not improve its accuracy, indicating that the OD's of false positive and

false negative results were not close to the selected cut-off value. In one of the two samples with a

false positive results, both the OroVax CagA recombinant based ELISA and H. pylori IgG-

antibodies were also false positive. The overall accuracy of the CagA rapid test (Orion) as

compared to the gold standard was 95.8%.

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124


125

Chapter 8

126

Discussion

Several studies have shown that anti-CagA antibodies can be detected in patients infected with

a cagA positive H. pylori strain [9-11,18]. The presence of anti-CagA antibodies correlates with

the presence of many cag pathogenicity island encoded virulence factors, and patients infected

with a cagA positive strain were shown to be more prone for the development of clinically

significant H. pylori-related disease. A significant association between the presence of cagA on the

one hand and PUD and gastric cancer on the other has been reported [19,20]. Moreover, a

recently published population based epidemiological study showed a significant association

between infection with a cagA positive strain and symptoms of dyspepsia [21]. On the other hand,

it has been suggested that infection with a cagA positive H. pylori strain protects against gastro-

esophageal reflux disease (GERD) and its consequences, Barrett’s metaplasia and adenocarcinoma

of the oesophagus [22,23]. As a result screening for anti-CagA antibodies might be useful to

identify patients with an increased risk for development of pathology, thus allowing selective

and/or differential treatment regimes [11,24]. A simple and reliable, non-invasive test for this

marker is thus not only a relevant tool for research purposes, but may also bear a clinical

relevance.

The relation between the presence of cagA and the clinical outcome of H. pylori infection is

primarily based on studies using the detection of anti-CagA antibodies to determine the cagA

status [20-22,25-27]. Only very few studies, however, (summarised in table 2) addressed the

accuracy of these serological tests [11,28-31]. The results of these studies are difficult to compare

as each study compared anti-CagA antibody serology to a different reference method, reflecting

the absence of a reliable generally accepted gold standard. Moreover, all studies only used a single

method as the gold standard, and all suffer from potential drawbacks. Firstly, the possibility of an

infection with both cagA positive and negative H. pylori strains has to be considered [32,33].

Secondly, it has been observed that H. pylori can spontaneously lose or obtain the cagA gene [34].

Thirdly, not all cagA positive H. pylori strains express CagA and cause an antibody response [11].

Finally, as in anti-H. pylori IgG serology, anti CagA-antibodies may persist after (accidental) H.

pylori eradication. Therefore, the reliability of PCR, colony hybridisation, or serology based

methods as the sole reference method is questionable.

In our study the agreement between PCR and the OroVax recombinant based ELISA was

compared and showed reasonable correspondence (83.7%). To avoid sampling error, we used the


127

whole biopsy specimen for our PCR. PCR showed considerably more cagA positive H. pylori

strains as compared to the OroVax recombinant based ELISA, possibly indicating the absence of

an anti-CagA antibody response in many patients. An absent immune response may be explained

by variations in the anti-body type between patients and the possibility that not all cagA positive

H. pylori strains express this gene. In only one patient the OroVax recombinant based ELISA

detected anti-CagA antibodies, while this patient was H. pylori negative by histology and culture.

In this patient, both anti-H. pylori IgG serology and the CagA rapid test (Orion) were also positive

which could point at sampling error when obtaining biopsy specimens or previous accidental

eradication of H. pylori with persisting circulating antibodies. Considering the troubling issues

hampering the formation of a gold standard, it is impossible to determine which of both tests, PCR

or OroVax recombinant based ELISA most reliably approaches reality. In this era of genome

based research, there is no longer a need to perform such studies with anti-CagA antibody serology

as the sole detection method. Therefore, we composed a gold standard based on those biopsy

specimens and serum samples in which both tests were in agreement. The CagA rapid test (Orion)

showed to be very accurate when compared to this gold standard. Moreover this test is easily

performed and no expensive laboratory equipment has to be used. Just a single drop of blood is

needed and the results can be read within ten minutes.

In conclusion, detection of H. pylori cagA by PCR and CagA antibody based ELISAs shows

reasonable agreement. The possibility of mixed infections, genetic drift, serological scars, and

cagA positive strains not expressing CagA make it impossible to determine which of these tests

approaches reality most reliably. When a gold standard was constructed based on concordance

between PCR and OroVax CagA recombinant based ELISA, the CagA rapid test (Orion) proved to

be very accurate.

Acknowledgements

The CagA rapid test (Orion) was supplied by Orion Diagnostica, Espoo, Finland

Chapter 8

128

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15. van Doorn LJ, Figueiredo C , Sanna R, Plaisier A, Schneeberger P, de Boer W et al. Clinical

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17. Loffeld RJ, Werdmuller BF, Kusters JG, Kuipers EJ . IgG antibody titer against Helicobacter pylori

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20. Parsonnet J, Friedman GD, Orentreich N, Vogelman H. Risk for gastric cancer in people with CagA

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26. Ching CK, Wong BC, Kwok E, Ong L, Covacci A, Lam SK. Prevalence of CagA-bearing

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27. Kikuchi S, Crabtree JE, Forman D, Kurosawa M. Association between infections with CagA-

positive or -negative strains of Helicobacter pylori and risk for gastric cancer in young adults. American


28. Yamaoka Y, Kodama T, Graham DY, Kashima K. Comparison of four serological tests to determine

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131

Chapter 9

Implications of the simultaneous presence of metronidazole-

susceptible and -resistant Helicobacter pylori colonies within a

single biopsy specimen.

Nicolaas L. A. Arents 1, Leonard C. Smeets 2, Anton A. van Zwet 1, Jacob C. Thijs 3,

Egbert Jan van der Wouden 3, Albertine de Jong 1, John E. Degener 4, and Johannes G.

Kusters 5.

1 Regional Public Health Laboratory, Groningen / Drenthe2 Department of Medical Microbiology, Free University, Amsterdam3 Department of Internal Medicine, Bethesda Hospital, Hoogeveen4 Department of Medical Microbiology, University Hospital, Groningen

Published in European Journal of Clinical Microbiology & Infectious Diseases

2001; 20: 418-420.

Chapter 9

132

Abstract

This study examined whether the simultaneous presence of metronidazole - susceptible and -

resistant Helicobacter pylori in a single biopsy specimen is caused by a multiple strain infection

with a susceptible and a resistant strain, or by two subpopulations within a single strain. Single

colonies obtained from seven biopsy specimens known to harbour both susceptible and resistant

Helicobacter pylori were fingerprinted by Restricted Fragment Length Polymorphism typing

(RFLP) of the ureC gene and by the Random Amplified Polymorphic DNA procedure (RAPD).

Metronidazole susceptibility was determined by E test. The results indicated that the occurrence of

metronidazole resistant and metronidazole susceptible bacteria within one single biopsy does not

imply the presence of a multiple strain infection with one resistant and one sensitive strain.

Introduction

Metronidazole is frequently used in the treatment of Helicobacter pylori infection.

Metronidazole resistance, however, is common and affects the efficacy of treatment regimens

containing this drug [1]. For that reason, culturing Helicobacter pylori for metronidazole

susceptibility testing has become mandatory. By such susceptibility testing several authors

observed that some patients are infected with both susceptible and resistant Helicobacter pylori

bacteria [2-5]. In a previous study we detected susceptible and resistant bacteria in 10% of our

biopsy specimens [3].

In 1989, another observation was made by Beji et al. [6]. These authors were the first to

describe the presence of more than one infecting strain in a single patient [6]. Since that time,

evidence for the occurrence of multiple Helicobacter pylori strain infection has accumulated and it

has been shown that this condition is not uncommon [7-10]. Both phenomena, heterogeneity in

metronidazole susceptibility and multiple strain infection, were extensively studied by Weel et al.

[4] and Dore et al. [5]. In both studies, Helicobacter pylori colonies obtained from biopsy

specimens known to harbour both metronidazole-susceptible and - resistant bacteria were

fingerprinted. Most biopsy specimens were colonised by a single strain, but about 10% contained

more than one Helicobacter pylori strain. The question whether these biopsy specimens were

infected with one susceptible Helicobacter pylori strain and one resistant strain, however, was not

answered as neither study mentioned any association between metronidazole susceptibility and

strain type. This study tries to address that question.

The simultaneous presence of metronidazole susceptible and resistant colonies

133

Materials and Methods

Seven biopsy specimens known to harbour both metronidazole-susceptible and metronidazole-

resistant Helicobacter pylori were studied. The biopsy specimens were obtained from separate

patients undergoing gastroduodenoscopy for dyspeptic symptoms. To our knowledge none of the

patients had previously received any anti-Helicobacter pylori treatment. Immediately after

endoscopy the biopsy specimens were transported in thyoglycolate broth to the laboratory and

used to inoculate Belo horizonte plates (BHM) containing brain heart infusion agar (35 mg/ml),

sheep blood (10%), vancomycin (6 mg/l), nalidixic acid (20 mg/l), amphotericin B (2 ml/l), and

2,3,5-triphenyltetrazolium chloride (40 mg/l). After incubation for 3 days under microaerobic

conditions, randomly selected single colonies were subcultured on separate BHM plates. The total

number of colonies selected from each primary culture plate varied between 8 and 12, depending

on the availability of clearly separated colonies. After another 3 days, these subcultures were used

to inoculate Columbia agar plates for metronidazole susceptibility testing as previously described

[3](E test; AB-Biodisk, Sweden) and BHM plates to produce stock colonies for DNA

fingerprinting. DNA isolation was performed according to the procedure described by Boom et al.

[11]. To fingerprint the Helicobacter pylori strains, we used two different procedures

recommended to type Helicobacter pylori: a Random Amplified Polymorphic DNA procedure

(RAPD) on the entire DNA and a Restricted Fragment Length Polymorphism typing (RFLP) of the

ureC gene [12,13]. RAPD-PCR reactions were performed in 50 microliter PCR reaction buffer,

containing 10 mM TRIS, 50 mM KCl, 3 mM MgCl2, 0.01% gelatine, 2 U Taq polymerase

(Boehringer-Mannheim, Germany), 0.25 mM dNTP's (Boehringer-Mannhein), 0.2 nmol primers

and 2 microliter isolated Helicobacter pylori DNA. The RAPD primers used were :

AAGAGCCCCG and : AACGCGCAAC. PCR was performed in a PTC 200 thermocycler (MJ

Research, USA) as follows: 9 min preincubation at 94°C, followed by 40 cycles of 30 s at 94°C,

45 s at 45°C and 45 s at 72°C. A final extension was performed for 10 min at 72°C. Twelve

microliter of the obtained product was used for gel electrophoresis, stained with ethidium bromide,

and photographed according to standard protocols. RFLP-PCR reactions were performed in 50

microliter PCR reaction buffer, containing 10 mM TRIS, 50 mM KCl, 3 mM MgCl2 , 0.01%

gelatine, 1.5 U Taq polymerase (Boehringer-Mannheim), 10 pmol dNTP's (Boehringer-

Mannheim), 40 pmol primers and 2 microliter isolated Helicobacter pylori DNA. PCR conditions

were 9 min preincubation at 94°C, followed by 35 cycles of 60 s at 94°C, 60 s at 50°C and 60 s at

Chapter 9

134

72°C. We used the primers described by Fujimoto et al. [9] for the amplification of the ureC gene,

'5-TGG GAC TGA TGG CGT GAG GG and '5-AAG GGC GTT TTT AGA TTT TT. These

primers yielded a 820 bp amplicon corresponding with the ureC gene. The amplicon was digested

by three different endonucleases according to the guidelines of the manufacturer (Boehringer-

Mannheim) : Sau 3A1, Tru 9I, and Cfo 1. The digested DNA was electrophoresed, stained with

ethidium bromide, and photographed according to standard protocols.

Results and Discussion

The MIC values for metronidazole for all tested colonies are shown in Table 1. As can be seen,

the selection of single colonies from each biopsy specimen enclosed both susceptible and resistant

Helicobacter pylori bacteria and a large variety of MIC values ranging from fully susceptible to

completely resistant was observed. Not all colonies tested for metronidazole susceptibility could

be fingerprinted successfully, as some colonies were lost in the process or DNA isolation failed. In

all biopsy specimens, however, fingerprinting results were available for both susceptible and

resistant colonies. Typing of the Helicobacter pylori colonies by both methods showed complete

concordance between RAPD and RFLP of the ureC gene. Fingerprinting showed a single

Helicobacter pylori strain in six of the seven biopsy specimens. In one biopsy specimen both

methods indicated the presence of two different Helicobacter pylori strains. Surprisingly, however,

the metronidazole resistant phenotype was not confined to a particular strain, as both Helicobacter

pylori strains were shown to consist of susceptible and resistant bacteria.

Our data show that in most biopsy specimens containing both susceptible and resistant

Helicobacter pylori bacteria, a single strain is involved. This confirms the finding of others,

showing a single strain infection in > 90% of the biopsy specimens with metronidazole susceptible

and resistant bacteria [4,5]. Weel et al. [4] found that in 28 of 156 consecutive patients, biopsy

specimens harboured both metronidazole - susceptible and - resistant Helicobacter pylori bacteria.

In 20 of these 28 specimens, single colonies were fingerprinted by RAPD and multiple strains

were found in two specimens (10%). Dore et al. [5] fingerprinted single colonies of 12 biopsy

specimens known to harbour Helicobacter pylori strains heterogeneous for metronidazole

susceptibility. Repetitive extragenic palindrome-based PCR typing showed that only one biopsy

specimen harboured two different, but closely related Helicobacter pylori strains (1 of 12). Neither

study, however, looked into the possibility that metronidazole resistance was confined to a


135

Chapter 9

136

particular strain. In our study, even in the biopsy specimen that contained two different

Helicobacter pylori strains, metronidazole susceptibility was not confined to a single strain as both

strains consisted of metronidazole - susceptible and -resistant bacteria.

It has been shown that in many cases, metronidazole resistance is caused by a null-mutation in

the rdxA gene, which encodes for a nitroreductase capable of converting metronidazole to its

active form [14-16]. Recently, another gene capable of reducing metronidazole has been described,

the frxA gene encoding for a NAD(P)H flavin oxidoreductase, which is possibly also involved in

metronidazole resistance among H. pylori isolates [17]. As heterogeneity in fmetronidazole

susceptibility is not explained by multiple strain infection, differences in susceptibility are more

likely caused by novel mutations in these genes within a single infecting strain.

References

1. van der Wouden EJ, Thijs JC , van Zwet AA, Sluiter WJ, Kleibeuker JH. The influence of in vitro

nitroimidazole resistance on the efficacy of nitromidazole containing anti-Helicobacter pylori regimens:

a meta-analysis. American Journal of Gastroenterology 1999; 94: 1751-1759.

2. Xia HHX, Kalantar J, Talley NJ. Metronidazole- and clarithromycin-resistant Helicobacter pylori in

dyspeptic patients in western Sydney as determined by testing multiple isolates from different gastric

sites. Journal of Gastroenterology and Hepatology 1998; 13: 1044-1049.

3. van der Wouden EJ, de Jong A, Thijs JC, Kleibeuker JH, van Zwet AA. Subpopulations of

Helicobacter pylori are responsible for discrepancies in the outcome of nitroimidazole susceptibility

testing. Antimicrobial Agents and Chemotherapy 1999; 43: 1484-1486.

4. Weel JF, van der Hulst RW, Gerrits Y, Tytgat GN, van der Ende A, Dankert J. Heterogeneity in

susceptibility to metronidazole among Helicobacter pylori isolates from patients with gastritis or peptic

ulcer disease. Journal of Clinical Microbiology 1996; 34: 2158-2162.

5. Dore MP, Osato MS, Kwon DH, Graham DY, El-Zaatari FAK. Demonstration of unexpected

antibiotic resistance of genotypically identical Helicobacter pylori isolates. Clinical Infectious Diseases

1998; 27: 84-89.

6. Beji A, Vincent P, Darchis I, Husson MO, Cortot A, Leclerc H. Evidence of gastritis with several

Helicobacter pylori strains. Lancet 1989; 9: 1402-1403.

7. van Doorn LJ, Figueiredo C, Rossau R, Jannes G, van Asbroeck M, Sousa JC et al. Typing of




137



9. Fujimoto S, Marshall B, Blaser MJ. PCR-based restriction fragment length polymorphism typing of

Helicobacter pylori. Journal of Clinical Microbiology 1994; 32: 331-334.

10. Hirschl AM, Richter M, Makristathis A, Pruckl PM, Willinger B, Schutze K et al. Single and

multiple strain colonization in patients with Helicobacter pylori-associated gastritis: detection by

macrorestriction DNA analysis. Journal of Infectious Diseases 1994; 170: 473-475.



12. Burucoa C, Lhomme V, Fauchere JL. Performance criteria of DNA fingerprinting methods for typing

Helicobacter pylori isolates: experimental results and meta-analysis. Journal of Clinical Microbiology

1999; 37: 4071-4080.

13. Lu JJ, Perng CL, Shyu RY, Chen CH, Lou Q, Chong SK et al. Comparison of five PCR methods for

detection of Helicobacter pylori DNA in gastric tissues. Journal of Clinical Microbiology 1999 ; 37:

772-774.

14. Goodwin A, Kersulyte D, Sisson G, Veldhuyzen van Zanten SJ, Berg DE, Hoffman PS .

Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an

oxygen-insensitive NADPH nitroreductase. Mol Microbiol 1998; 28: 383-93.

15. Hoffman PS, Goodwin A, Johnsen J, Magee K, Veldhuyzen van Zanten SJ. Metabolic activities of

metronidazole-sensitive and -resistant strains of Helicobacter pylori: repression of pyruvate

oxidoreductase and expression of isocitrate lyase activity correlate with resistance. Journal of

Bacteriology 1996; 178: 4822-4829.

16. Jenks PJ, Ferrero RL, Labigne A. The role of the rdxA gene in the evolution of metronidazole

resistance in Helicobacter pylori. Journal of Antimicrobial Chemotherapy 1999; 43: 753-758.

17. Kwon DH, Kato M, El Zaatari FA, Osato MS, Graham DY. Frame-shift mutations in NAD(P)H

flavin oxidoreductase encoding gene (frxA) from metronidazole resistant Helicobacter pylori

ATCC43504 and its involvement in metronidazole resistance. FEMS Microbiology Letters 2000; 188:

197-202.

139

Chapter 10

Summary and general conclusions.

Chapter 10

140

Summary and conclusions

In this thesis the management of dyspepsia in primary care in relation to Helicobacter pylori

(H. pylori) infection was studied. In chapter two several important issues considering the approach

of dyspepsia are discussed. It is clear that dyspepsia is not a disease but merely a cluster of

symptoms believed to originate from the gastrointestinal tract. This is probably the main reason

why a universal consensus regarding the approach of dyspepsia is still lacking. Although many

studies have evaluated strategies for dyspepsia, considerable differences exist in the definitions of

dyspepsia, hampering a reliable comparison between study results. The discovery of H. pylori has

resulted in new strategies to approach dyspepsia in primary care, based on screening for H. pylori

infection. Altogether, four main strategies for dyspepsia are currently considered: 1. Symptom

guided empirical treatment, 2. Direct referral for endoscopy, 3. Non-invasive testing for H. pylori

and subjecting the H. pylori-positive patients to endoscopy (“test-and-scope”), and 4. Non-

invasive testing for H. pylori and treatment of the infection in H. pylori-positive patients (“test-

and-treat”). Based on data derived from the literature, several key issues were identified which

have a significant impact on the cost-effectiveness of these strategies, namely: cost of endoscopy,

cost of therapy, cost of physician visit, prevalence of H. pylori in the dyspeptic population,

prevalence of peptic ulcer disease (PUD) in H. pylori positive patients, prevalence of all

underlying diseases in dyspepsia, benefit of H. pylori eradication in patients with functional

dyspepsia, and recurrence of dyspeptic symptoms in patients with (functional) dyspepsia after

treatment. After careful consideration of the literature with respect to the advantages and

disadvantages of the various strategies and the key issues mentioned above, it is concluded that at

this moment, a “test-and-treat” strategy is the most rational approach in the younger patient with

uncomplicated dyspepsia.

Chapter three reports the results of the SENSE study (Strategy: ENdoscopy versus SErolgy).

Patients who visited their general practitioner (GP) for dyspepsia could participate in the study if

the GP judged the symptoms severe enough to justify treatment with acid-suppressive drugs or an

upper endoscopy. Two hundered seventy patients fulfilling this condition were randomised to

either a serology based “test-and-treat” strategy or a prompt open-access endoscopy. The study

was performed completely in a primary care setting in the northern part of the Netherlands. After a

one year follow-up period, only 33% of the patients in the “test-and-treat” arm were eventually

referred for endoscopy. In these patients, no H. pylori associated PUD was detected. Symptom

Summary and general conclusions

141

resolution, change in quality of life, and patient satisfaction were comparable between the “test-

and-treat” patients and the patients in the prompt endoscopy arm. The “test-and-treat” patients,

however, visited their GP more frequently and were more frequently prescribed prokinetic drugs.

On the other hand, prompt endoscopy patients were more often prescribed PPI's. Altogether, the

SENSE study proved that the “test-and-treat” strategy is a save and cost-effective approach in

primary care.

Before a “test-and-treat” strategy can be savely implemented in primary care, however, several

conditions need to be fulfilled.

Firstly, reliable non-invasive testing for H. pylori has to be possible and therefore, accurate

tests are required. In chapter four, the accuracy of the recently introduced Helicobacter pylori

Stool Antigen (HpSA) test, which is based on the detection of H. pylori antigens in faecal samples

by antigen capture ELISA, was evaluated. This test showed accurate results for the detection of H.

pylori infection in untreated patients (sensitivity and specificity of 96.3% and 81.8% respectively)

when compared to a gold standard based on culture, histology, and rapid-urease testing of gastric

biopsy specimens. Both one and four weeks after cessation of therapy, however, the test failed to

detect persisting H. pylori infection (sensitivity 20.0%, specificity 95.0% (at one week), sensitivity

40.0% and specificity 95.0% (at four weeks)). These results are in contrast to the results published

by others. No adequate explanation can be given for this discrepancy. It is concluded, however,

that the HpSA test is not sufficiently reliable to monitor treatment success, at least in our hands.

Secondly, as stated before in chapter two, the cost-effectiveness of strategies based on

screening for H. pylori mainly depend on the number of patients who will clearly benefit from

anti-H. pylori therapy. As these are patients suffering from H. pylori related PUD, a decrease in

the prevalence of H. pylori related PUD will result in a decreased cost-effectiveness of the “test-

and-treat” strategy. Recently, it has been suggested that the significance of H. pylori in PUD has

been overestimated and that the decrease in H. pylori prevalence will unmask idiopathic PUD (not

related to NSAID use or H. pylori infection). In chapter five the results of all endoscopies

performed over an eigth year period in a single hospital were studied. These data revealed a

decreasing trend in the prevalence of H. pylori in PUD. The proportion of PUD ascribed to

NSAID-use increased significantly, but the proportion ascribed to idiopathic PUD remained stable

at about 10% during the study period.

Chapter 10

142

Thirdly, a “test-and-treat” strategy in primary care is only useful if patients can be effectively

treated in the absence of information on antibiotic susceptibility. Numerous studies have shown

that antibiotic resistance has a significant impact on the efficacy of anti-H. pylori regimens. In the

Netherlands, the most frequently used anti-H. pylori regimens are the triple regimens PAM

(protonpump inhibitor, amoxicillin, metronidazole), and PAC (protonpump inhibitor, amoxicillin,

clarithromycin). In chapter six we showed that in the north-eastern part of the Netherlands the

prevalence of metronidazole resistance decreased significantly from 1996 to 2001. The prevalence

of clarithromycin resistance fluctuated over this period, but no significant trend could be observed.

Interestingly, the decrease in prevalence of metronidazole resistance was accompanied by a

concurrent decrease in the number of metronidazole prescriptions in the Netherlands (precriptions

for other conditions than anti-H. pylori treatment were also included). Altogether both regimens

(PAM and PAC) had a calculated efficacy of > 80%. The efficacy of the PAM regimen is likely to

increase if the decrease in prevalence of metronidazole resistance continues, whereas the efficacy

of the PAC regimen will drop significantly after even a slight increase in the prevalence of

clarithromycin resistance.

The second part of the thesis focussed on genetic differences between H. pylori strains. These

genetic differences have been hold responsible for the differences in clinical outcome. Several

virulence factors have been identified in H. pylori, like the vacA gene which encodes for the

vacuolating cytotoxin, the cagA gene responsible for the production of the CagA protein, and the

iceA gene, which is expressed at contact with gastric epithelial cells. In chapter seven, we have

studied the association between these virulence factors and various clinical outcomes (PUD, gastro

oesophageal reflux disease (GERD) and gastritis only). The results of this study showed that many

patients (over 20%) harboured a mixture of H. pylori strains. PUD patients were most frequently

infected with an H. pylori strain of the s1 vacA genotype or a strain which carried the cagA gene.

On the other hand, patients with GERD, were most frequently colonized by a strain lacking both

the cagA and the iceA1 gene. This finding supports the hypothesis that more virulent H. pylori

strains may protect against the development of GERD. It was concluded that virulence factors are

indeed associated with specific clinical outcomes. Whether the detection of these virulence factors

is significant in a clinical setting, however, needs further exploration. If so, a reliable non-invasive

test to detect the presence of these virulence factors in dyspeptic patients would be essential.

Chapter eight reports the results of a study evaluating the accuracy of the CagA rapid test (Orion).

Summary and general conclusions

143

It has been claimed that this test enables the accurate detection of CagA-antibodies in serum

samples by an easy to perform immunochromatographic test, showing results within 10 minutes.

We compared the CagA rapid test (Orion) to a surrogate gold standard based on a combination of

PCR based cagA gene detection in gastric biopsies and the OroVax recombinant based ELISA for

the detection of serum antibodies. The gold standard was considered positive if both PCR and

OroVax recombinant based ELISA were positive. In other cases, the gold standard was considered

negative. This gold standard was chosen, as it is well known that the presence of the cagA gene

does not always result in the transcription of this gene. Without transcription serological antibodies

may be absent. On the other hand, persisting CagA-antibodies after a previously cleared H. pylori

infection may result in a positive CagA rapid test (Orion), whereas the PCR results are negative.

Compared to the surrogate gold standard, the CagA rapid test (Orion) showed a sensitivity and a

specificity of 96.8% and 95.4% respectively. Finally, in chapter nine the results are reported of an

extensive study performed on biopsy specimens of nine patients in whom both metronidazole

susceptible and resistant H. pylori bacteria had been demonstrated. The question was whether

these patients were colonized by two different H. pylori strains or by a single strain, part of which

became resistant. The second hypothesis appeared to be true as eight patients harboured only a

single H. pylori strain, part of which was sensitive and part of which was resistant to

metronidazole. In one patient, the presence of two different strains could be demonstrated, but both

strains were in part susceptible and resistent to metronidazole. Based on these data it is concluded

that the occurence of metronidazole resistant and metronidazole susceptible bacteria within one

single biopsy specimen does not imply the presence of a multiple strain infection with one

resistant and one susceptible strain.

General conclusions

The discovery of the role of H. pylori in several diseases of the upper alimentary tract has

resulted in new strategies to approach uncomplicated dyspepsia. Several authors have suggested

that an approach based on screening for H. pylori may be more rational than the common strategy

of empirical treatment with acid-suppressive drugs. This suggestion is supported by both decision

analytic studies and clinical studies, among which the SENSE study described in this thesis.

Moreover, most studies suggest that such an approach, especially the “test-and-treat” strategy, may

be more cost-effective. In contrast to most other clinical studies, the SENSE study was completely

Chapter 10

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performed in a primary care setting. It showed that in primary care a “test-and-treat” strategy is

safe, is more cost-effective than a prompt endoscopy approach, and results in comparable

symptom resolution.

Implementation of the “test-and-treat” strategy in primary care is feasible, as accurate non-

invasive tests to diagnose H. pylori infection are available. Moreover, given the current levels of

antibiotic resistance, treatment of H. pylori infection with the usual PAC or PAM regimens should

result in acceptable success rates (>80%), even in the abscence of information on antibiotic

susceptibility. Monitoring of antibiotic resistance in H. pylori, however, remains mandatory, as

even a slight increase in the prevalence of clarithromycin resistance will result in a sharp drop in

cure rates if the PAC regimen is used.

Any strategy for dyspepsia that is based on non-invasive detection of H. pylori is critically

dependent on the prevalence of H. pylori in the dyspeptic population. At a low prevalence, the

positive predicitive value of any test for H. pylori will drop below an acceptable level, prohibiting

the implementation of a “test-and-treat” strategy. Moreover, if the prevalence of H. pylori in the

dyspeptic population is to low, conditions unrelated to H. pylori infection (among which idiopathic

PUD) are more likely to be the cause of dyspepsia. In our view, however, the current prevalence of

H. pylori in the dyspeptic population (>30% in the SENSE study) still justifies a “test-and-treat”

approach of uncomplicated dyspepsia in primary care. It is feasible, safe and cost-effective. The

prevalence of H. pylori infection, however, is rapidly decreasing in the Western world, and it is

likely that empirical treatment with acid suppression will eventually become more cost-effective

than the “test-and-treat” strategy. As the prevalence of H. pylori is strongly associated with age

this may already be true for the very young dyspeptic patient.

The virulence of the infecting H. pylori strain is probably related to the clinical outcome of the

infection. However, as the most virulent strains at the same time seem to cause PUD and protect

against GERD, it is unlikely that the non-invasive detection of virulence factors will contribute to

the clinical decision to treat or not to treat an infected patient, even though accurate tests are

available. Finally, it should be stressed that prompt endoscopy is the only acceptable approach of

any patient presenting with alarm symptoms.

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Chapter 11

Samenvatting en algemene conclusies.

146

Samenvatting en algemene conclusies

Dit proefschrift beschrijft het onderzoek naar de benadering van dyspepsie in de

huisartspraktijk in relatie tot een infectie met Helicobacter pylori (H. pylori). In hoofdstuk twee

worden enkele belangrijke punten besproken voor wat betreft de benadering van dyspepsie. Het is

duidelijk dat dyspepsie geen specifieke aandoening is, maar eerder een verzameling van

symptomen waarvan gedacht wordt dat ze ontstaan vanuit het bovenste deel van het

maagdarmkanaal. Dit is waarschijnlijk de belangrijkste reden waarom er nog steeds geen algemene

overeenstemming bestaat over de benadering van dyspepsie. Hoewel veel studies verschillende

benaderingsstrategieën voor dyspepsie hebben onderzocht, zijn er grote verschillen in de gebruikte

definitie van dyspepsie, het geen een betrouwbare vergelijking van de verschillende studies

bemoeilijkt. De ontdekking van H. pylori heeft nieuwe strategieën voor de benadering van

dyspepsie in de huisartspraktijk mogelijk gemaakt die gebaseerd zijn op screening voor de

aanwezigheid van een H. pylori infectie. De belangrijkste strategieën op dit moment zijn : 1. Een

proefbehandeling op geleide van de klachten, 2. Direkte verwijzing voor een gastroduodenoscopie,

3. Niet-invasief testen voor H. pylori infectie waarbij alleen de positieve patiënten direkt worden

verwezen voor een gastroduodenoscopie (“test-and-scope”), en 4. Niet-invasief testen voor H.

pylori infectie waarbij positieve patiënten direkt een anti-H. pylori behandeling krijgen

voorgeschreven (“test-and-treat”). Uit de literatuur over dit onderwerp blijkt dat enkele factoren

een sleutelrol spelen voor wat betreft de kosten-effectiviteit van de verschillende strategieën,

namelijk : de kosten van een gastroduodenoscopie, de kosten van een behandeling, de kosten van

een huisartsconsult, de prevalentie van H. pylori infectie in dyspeptische patiënten, de prevalentie

van peptische ulcera in H. pylori positieve patiënten, de prevalentie van de verschillende

aandoeningen die dyspepsie kunnen veroorzaken, het nut van H. pylori eradicatie in patiënten met

functionele dyspepsie, en het aantal patiënten met recidiverende (functionele) klachten na

behandeling. Na zorgvuldige bestudering van de gegevens uit de literatuur en de genoemde

sleutelfactoren is de conclusie dat op dit moment een “test-and-treat” strategie de meest rationele

benadering is voor patiënten met dyspepsie in de huisartspraktijk.

In hoofdstuk drie worden de resultaten van de SENSE studie (Strategy : ENdoscopy versus

SErology) beschreven. Patiënten die hun huisarts bezochten wegens dyspeptische klachten konden

aan de studie deelnemen indien de huisarts de klachten ernstig genoeg vond om een

proefbehandeling met zuurremmers te starten of de patiënt voor een gastroduodenoscopie te

Sammenvatting en algemene conclusies

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verwijzen. Tweehonderdzeventig patiënten die aan deze criteria voldeden zijn uiteindelijk

gerandomiseerd in een groep die volgens de “test-and-treat” strategie werd benaderd en een groep

die direkt voor een gastroduodenoscopie werd verwezen (“direkte scopie”). De studie werd

volledig in Noord-Nederlandse huisartspraktijken uitgevoerd. Na een vervolgperiode van één jaar

bleek dat slechts 33% van de patiënten in de “test-and-treat” groep alsnog voor een

gastroduodenoscopie was verwezen. Tijdens de gastroduodenoscopie werd in geen van deze

patiënten een H. pylori gerelateerd peptisch ulcus gevonden. Na één jaar was de mate van het

verdwijnen van de dyspeptische symptomen, de verandering in kwaliteit van leven en de

tevredenheid van de patiënten vergelijkbaar in beide studiegroepen. De patiënten in de “test-and-

treat” groep bezochten significant vaker de huisarts tijdens de studieperiode en kregen vaker

prokinetica voorgeschreven. Aan de andere kant kregen patiënten in de “direkte scopie” groep

vaker protonpompremmers voorgeschreven. Samengevat heeft de SENSE studie laten zien dat een

“test-and-treat” strategie veilig en kosten-effectief kan zijn voor de benadering van patiënten met

dyspepsie in de huisartspraktijk.

Voordat een “test-and-treat” strategie veilig en probleemloos kan worden ingevoerd in de

huisartspraktijk moet aan enkele voorwaarden zijn voldaan.

Allereerst moet het mogelijk zijn een H. pylori infectie door middel van niet-invasieve

methoden aan te tonen, waarvoor betrouwbare H. pylori testen noodzakelijk zijn. In hoofdstuk vier

wordt de recent geintroduceerde Helicobacter pylori Stool Antigeen test (HpSA test), die

gebaseerd is op het aantonen van H. pylori antigenen in de ontlasting door middel van “stool

capture ELISA”, geëvalueerd. Het bleek dat deze nieuwe test ten opzichte van de gouden

standaard die gebaseerd was op het testen van maagbiopten door middel van kweek, histologie en

ureasetest, betrouwbaar de aanwezigheid van een H. pylori infectie detecteerde in patiënten die

nooit eerder behandeld waren voor deze infectie (sensitiviteit en specificiteit van respectivelijk

96.3% en 81.8%). De HpSA test bleek niet erg betrouwbaar een persisterende H. pylori infectie te

detecteren in patiënten die één of vier weken eerder een anti-H. pylori behandeling hadden

voltooid (sensitiviteit en specificiteit respectievelijk 20.0% en 95.0% (na één week) en 40.0% en

95.0% (na vier weken). Deze resultaten komen niet overeen met de resultaten die door anderen

gepubliceerd zijn. Daarvoor is geen goede verklaring te geven. Het is echter onze mening, dat op

dit moment de HpSA test onvoldoende betrouwbaar is om in onze patiëntenpopulatie het succes

van een anti-H. pylori behandeling te controleren.

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Ten tweede, zoals al eerder aangegeven in hoofdstuk twee, hangt de kosten-effectiviteit van

strategieën die gebaseerd zijn op screening voor H. pylori sterk af van het aantal patiënten dat

gunstig reageert op anti-H. pylori behandeling. Omdat dit meestal patiënten zullen zijn met een

peptisch ulcus, zal een afname van het aantal patiënten met een H. pylori gerelateerd peptisch

ulcus leiden tot een lagere kosten-effectiviteit van de “test-and-treat” strategie. Kort geleden is

gesuggereerd dat de rol van H. pylori bij peptische ulcera overschat wordt en dat de afnemende

prevalentie van H. pylori infecties kan leiden tot een ontmaskering van idiopathische peptische

ulcera (waarbij een H. pylori infectie noch NSAID gebruik aantoonbaar zijn). In hoofdstuk vijf

worden de resultaten beschreven van een onderzoek naar de bevindingen van alle gastro-

duodenoscopiën die verricht zijn tijdens een acht jaar durende periode in het Bethesda ziekenhuis

in Hoogeveen. Deze data laten inderdaad zien dat er een significante afnemende tendens is van het

aantal H. pylori geassocieerde peptische ulcera. De trend ging gepaard met een toenemend aantal

NSAID-gerelateerde peptische ulcera. De prevalentie van idiopathische ulcera bleef ongeveer

gelijk op 10% gedurende de studie periode.

Ten derde, een “test-and-treat” strategie in de huisartspraktijk kan alleen succesvol uitgevoerd

worden indien er tevens een effectieve behandelingsmogelijkheid is, zelfs als er geen informatie

beschikbaar is over de gevoeligheid van de H. pylori stam. Veel studies hebben aangetoond dat

resistentie tegen antibiotica een significante negatieve invloed heeft op de effectiviteit van anti-H.

pylori behandelingen. De meest voorgeschreven anti-H. pylori behandelingen in Nederland zijn de

tripel-behandelingen PAM (een combinatie van een protonpompremmer, amoxicilline en

metronidazol) en PAC (een combinatie van een protonpompremmer, amoxicilline en

clarithromycine). Hoofdstuk zes laat zien dat in Noord-Nederland de prevalentie van

metronidazol-resistentie siginificant daalde gedurende de periode 1996 - 2001. De prevalentie van

clarithromycine-resistentie fluctueerde gedurende deze periode, zonder dat er een trend

aantoonbaar was. Het was opvallend dat de dalende tendens in de prevalentie van metronidazol-

resistentie samen viel met een afname in het aantal voorschriften voor metronidazol in Nederland

(inclusief metronidazolvoorschriften voor andere aandoeningen dan H. pylori infectie).

Samengevat hadden beide tripel-behandelingen (PAM en PAC) een berekende effectiviteit van

> 80%. Het is aannemelijk dat de effectiviteit van de PAM-behandeling verder zal toenemen

indien de prevalentie van metronidazol-resistentie verder zal dalen. Daarentegen zal bij een


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geringe toename van de prevalentie van clarithromycine-resistentie de effectiviteit van de PAC-

behandeling onder de 80% zakken.

Het tweede deel van dit proefschrift richt zich met name op genetische verschillen tussen

H. pylori stammen. Deze genetische verschillen worden mede verantwoordelijk gehouden voor de

verschillen in symptomen en ziektebeelden die veroorzaakt kunnen worden door een een H. pylori

infectie. Verschillende virulentiefactoren van H. pylori zijn aangetoond, zoals het vacA gen wat

codeert voor het vacuolating cytotoxine, het cagA gen wat verantwoordelijk is voor de produktie

van het CagA eiwit, en het iceA gen dat tot expressie komt na contact met maagepitheelcellen. In

hoofdstuk zeven hebben wij het verband tussen deze virulentiefactoren en de verschillende

ziektebeelden bestudeerd (peptisch ulcus, refluxziekte en maagslijmvliesontsteking). De resultaten

van deze studie lieten zien dat een groot gedeelte van de patiënten (meer dan 20%) geinfecteerd is

met meerdere H. pylori stammen. Patiënten met een peptisch ulcus bleken meestal geinfecteerd te

zijn door een H. pylori stam met het s1 vacA genotype of een stam dat het cagA gen bezat. Aan de

andere kant waren patiënten met refluxziekte meestal geinfecteerd met een H. pylori stam waarin

zowel het cagA als het iceA1 gen afwezig waren Deze bevinding ondersteunt de hypothese dat

virulente H. pylori stammen beschermen tegen het ontwikkelen van refluxziekte. Concluderend

kan gezegd worden dat virulentiefactoren van H. pylori inderdaad geassocieerd zijn met het

uiteindelijke ziektebeeld. Of deze virulentiefactoren ook gebruikt kunnen worden in de dagelijkse

klinische praktijk moet nog verder onderzocht worden. Een betrouwbare niet-invasieve methode

om de aanwezigheid van verschillende virulentiefactoren aan te tonen zou een nuttige aanvulling

zijn om de associatie tussen virulentiefactoren en ziektebeelden verder te bestuderen. Hoofdstuk

acht beschrijft de resultaten van een studie die de betrouwbaarheid van de CagA rapid test (Orion)

evalueerde. Deze test zou op betrouwbare wijze binnen tien minuten door middel van een

immuunchromatografische test de aanwezigheid van antilichamen tegen het CagA eiwit in

serummonsters kunnen detecteren. Wij hebben de CagA rapid test (Orion) vergeleken met een

surrogaat gouden standaard gebaseerd op een combinatie van een PCR op het cagA gen in

maagbiopten en de OroVax recombinant test, gebaseerd op het aantonen van antilichamen in

serum door middel van de ELISA techniek. De gouden standaard werd als positief beschouwd

indien zowel de cagA PCR als de OroVax recombinant test positief waren. Andere combinaties

werden als negatief beschouwd. Deze gouden standaard werd gekozen omdat het bekend is dat de

aanwezigheid van het cagA gen niet altijd betekent dat dit gen ook tot expressie komt. Zonder

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expressie van het gen zal er ook geen immuunrespons op gang komen. Aan de andere kant kunnen

antilichamen tegen het CagA eiwit blijven circuleren ondanks een succesvol behandelde infectie.

Hierdoor kunnen de CagA rapid test (Orion) en de OroVax recombinant test positief blijven,

terwijl de cagA PCR negatief blijkt. In vergelijking met de surrogaat gouden standaard toonde de

CagA rapid test (Orion) een sensitiviteit en specificiteit van respectievelijk 96.8% en 95.4%. Als

laatste worden in hoofdstuk negen de resultaten gepresenteerd van een studie op negen biopten van

negen patiënten die een H. pylori infectie bleken te hebben met zowel metronidazol gevoelige als

resistente H. pylori bacteriën. De vraag was of deze patiënten geinfecteerd waren met twee

verschillende H. pylori stammen of dat ze geinfecteerd waren met één enkele H. pylori stam

waarvan een gedeelte resistent was geworden tegen metronidazol. De tweede stelling bleek waar te

zijn aangezien acht van de negen patiënten één enkele H. pylori stam bij zich droegen, waarvan

een gedeelte resistent was tegen metronidazol. De overgebleven patiënt bleek gekoloniseerd te zijn

met twee verschillende H. pylori stammen die beiden deels gevoelig en deels resistent waren tegen

metronidazol. Uitgaande van deze resultaten werd geconcludeerd dat de aanwezigheid van zowel

metronidazol gevoelige als resistente H. pylori fenotypen in één patiënt niet automatisch betekent

dat deze patiënt ook geinfecteerd is met twee verschillende H. pylori stammen waarvan er één

gevoelig is voor metronidazol en de andere resistent is voor metronidazol.

Algemene conclusies

De ontdekking van de belangrijke rol van H. pylori in verschillende aandoeningen van de

bovenste tractus digestivus heeft geleid tot nieuwe benaderingsmogelijkheden van de patiënt met

ongecompliceerde dyspepsie in de huisartspraktijk. Enkele auteurs hebben gesuggereerd dat een

benadering gebaseerd op screening voor H. pylori waarschijnlijk rationeler is dan de huidige

strategie bestaande uit een proefbehandeling met zuurremmers. Deze suggestie wordt ondersteund

door zowel analytische studies als door klinische studies, waaronder de SENSE studie die in dit

proefschrift wordt beschreven. Daarnaast blijkt uit de meeste studies dat een screeningsstrategie,

en met name de “test-and-treat” strategie, mogelijk meer kosten-effectief is. In tegenstelling tot de

meeste andere studies is de SENSE studie volledig in de eerste lijn uitgevoerd. Uit de SENSE

studie is gebleken dat een “test-and-treat” strategie veilig is, meer kosten-effectief is dan, en tot

een zelfde mate van reductie van symptomen leidt als een strategie waarbij alle patiënten direkt

voor een gastroduodenoscopie worden verwezen.


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Het toepassen van een “test-and-treat” strategie in de huisartspraktijk is mogelijk aangezien er

betrouwbare, niet-invasieve testen beschikbaar zijn om een H. pylori infectie aan te tonen.

Daarnaast is gebleken dat gezien de huidige prevalenties van antibioticaresistentie in H. pylori een

behandeling met de meest voorgeschreven kuren (PAM en PAC tripel-kuren) in meer dan 80%

van de patiënten leidt tot eradicatie van H. pylori. Het controleren van de prevalentie van

antibioticaresistentie blijft echter noodzakelijk omdat slechts een kleine toename in de prevalentie

van clarithromycine-resistentie al leidt tot een aanzienlijke reductie van de effectiviteit van de

PAC behandeling.

De effectiviteit van iedere H. pylori screeningsstrategie voor de benadering van dyspepsie is

grotendeels afhankelijk van de prevalentie van H. pylori in de populatie. Bij een lage prevalentie

wordt de positief voorspellende waarde onacceptabel laag, waardoor een “test-and-treat” strategie

praktisch niet meer uitvoerbaar is. Daarnaast zullen andere aandoeningen relatief vaker de oorzaak

van de klachten worden indien de prevalentie van H. pylori infectie in dyspeptische patiënten daalt

(waaronder onder andere idiopathische peptische ulcera). Het mag evenwel gesteld worden dat de

huidige prevalentie van H. pylori infectie (>30% in de SENSE studie) een “test-and-treat” strategie

rechtvaardigt voor de benadering van patiënten met ongecompliceerde dyspepsie in de

huisartspraktijk. Deze strategie blijkt uitvoerbaar en is kosten-effectief. De prevalentie van

H. pylori infectie is echter sterk aan het dalen in de Westerse wereld en het is waarschijnlijk dat

een proefbehandeling met zuurremmers uiteindelijk kosten-effectiever wordt dan een “test-and-

treat” strategie. De kans op een H. pylori infectie is sterk afhankelijk van de leeftijd van de patiënt

en neemt toe naarmate de patiënt ouder is. Daarom is een proefbehandeling met zuurremmers voor

zeer jonge patiënten met dyspepsie waarschijnlijk al kosten-effectiever dan een “test-and-treat”

strategie.

Het ziektebeeld dat een H. pylori infectie veroorzaakt is afhankelijk van de virulentie van de H.

pylori stam. Gezien het feit dat virulente H. pylori stammen enerzijds geassocieerd zijn met

peptische ulcera, maar anderzijds mogelijk een beschermende rol spelen tegen refluxziekte, is het

onwaarschijnlijk dat niet-invasieve testen voor het aantonen van virulentiefactoren een

doorslaggevende rol zullen gaan spelen in de beslissing een patiënt wel of niet te behandelen. Als

laatste blijft het belangrijk te benadrukken dat direkte verwijzing voor gastroduodenoscopie de

enige goede benadering blijft voor dyspeptische patiënten die zich presenteren met

alarmsymptomen.

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Dankwoord

Promo-veren zijn de veren van een ander waarmee een promovendus een dagje mag pronken.

Zo zou eigenlijk de omschrijving moeten luiden van het begrip “promoveren”. Hoewel ik erg trots

ben op het werkje dat hier nu voor u ligt besef ik mij terdege dat ik dit resultaat nooit bereikt zou

kunnen hebben zonder de inspanning van vele anderen.

Allereerst wil ik Jaap Thijs en Ton van Zwet bedanken. Vele jaren geleden hebben zij zich al

vastgebeten in Helicobacter pylori. Het onderzoek dat zij destijds verrichtten vormt nu de basis

voor alweer het derde proefschrift in de reeks Thijs, van Zwet & Helicobacter pylori. Jaap, jouw

heldere manier van redeneren en formuleren beschouw ik nog steeds als de “gouden standaard”.

Hoewel jouw nauwkeurige en perfectionistische instelling destijds niet altijd in dank werd

afgenomen, ben ik langzaam aan in gaan zien dat dit de enige correcte houding is om wetenschap

te beoefenen. Hopelijk kan ik deze standaard vasthouden. Ton, jij symboliseerde de “Yan”-helft

van de co-promotoren. Jouw enthousiasme en optimisme om nieuwe wegen in te slaan en jouw

vermogen om kogels door kerken te schieten en schepen achter ons te verbranden heeft ertoe

geleid dat we niet nog steeds met het eerste artikel bezig zijn, maar het uiteindelijk tot acht

artikelen hebben geschopt ! Hopelijk heb ik een deel van jouw optimisme en besluitvaardigheid

over kunnen nemen.

Tevens wil ik prof. Kleibeuker bedanken. Als na veel wikken en wegen in Hoogeveen besloten

werd dat een artikel gesubmit kon gaan worden was het altijd prettig dat u de laatste puntjes op de

i kon zetten. Ook op de momenten dat ik eindeloos met kleine details worstelde wist u mij weer de

grote lijnen te laten zien.

Prof. Degener, mijn andere promotor, wil ik ook graag bedanken. Hoewel het proefschrift met

name gebaseerd is op gastroenterologische aspecten van Helicobacter pylori en dyspepsie waren

uw adviezen vanuit de microbiologische hoek zeer waardevol.

Ook Albertine de Jong ben ik veel dank verschuldigd. Albertine, vooral in de eerste jaren van

mijn promotieonderzoek heb jij ontelbare Helicobacter kweken verricht en PCRs gedraaid. Nooit

was jij te beroerd om weer met een portie maagbiopten naar het Streeklaboratorium in Groningen

te reizen om ze vervolgens binnenste buiten te keren op zoek naar de Helicobactertjes. Langzaam

leek het Helicobacter pylori project ook de hele research afdeling van het Streeklaboratorium te

besmetten. Met name Mirjam Kooistra-Smid en Cees van Slochteren hebben meerdere steentjes

bijgedragen aan dit proefschrift, waarvoor mijn dank. Enkele analisten van de dependance van het

154

Streeklaboratorium in Hoogeveen wil ik ook graag met name noemen. Joseph Nanuru, Djoeke

Kelly, Marriëtte Borggreve en Corrie Borger hartelijk dank voor het zo nauwkeurig bijhouden en

bewaken van de Helicobacter pylori isolaten.

Verder wil ik Egbert-Jan van der Wouden bedanken. Tijdens de eerste twee jaar van mijn

promotie, waarbij wij op één kamer zaten, heb jij mij het nodige over Helicobacter geleerd.

Achteraf gezien ben ik ervan overtuigd dat het rendement van onze langdurige discussies over

Helicobacter daarna nooit meer overtroffen is door de vele uren zelfstudie. Ook Michiel Wulfelle

heeft een belangrijke bijdrage geleverd aan het plezierige gevoel dat ik krijg als ik aan de

“Hoogeveense” periode terug denk. Samen met Egbert-Jan hebben wij regelmatig in Hoogeveen

en Groningen de wetenschap, het darwinisme, de politiek, en andere onzin bediscussiëerd onder

het genot van een koud glas “Murphy's Red”. En dan heb ik het nog niet eens over de vele

“game”-uren waarin we tijdelijk andere organismen op de korrel namen.

Dank ben ik ook verschuldigd aan Ingrid Barkema, de secretaresse van Jaap Thijs. Jouw

vrolijke instelling en behulpzaamheid heeft onze samenwerking altijd erg plezierig gemaakt.

Daarnaast wil ik alle andere secretaresses van de Interne bedanken met name Joma Kaal, Janke

Warnders, Marike Kats en Ineke Zandvliet. Na enkele uren Helicobacter artikelen kauwen was het

altijd erg fijn om weer eens op een gewone boterham te kauwen en te horen dat er nog meer in het

leven is dan Helicobacter pylori.

De SENSE studie was nooit mogelijk geweest zonder de 56 huisartsen die bereid waren een

deel van hun tijd en patiënten te investeren in onze studie. Het moet erg moeilijk geweest zijn om

patiënten bereid te vinden mee te doen aan een studie waarbij zij kans hebben een maagonderzoek

te ondergaan. Desalniettemin is het uiteindelijk toch gelukt 291 patiënten in te sluiten. Hartelijk

dank voor deze enorme prestatie ! Ook de gastroenterologen Marco Oudkerk Pool en Jan Mark

Götz hebben een belangrijke bijdrage geleverd aan de SENSE studie. Daarnaast wisten zij mij

altijd weer een hart onder de riem te steken in tijden dat de inclusie moeizaam verliep.

Aventis Pharma, met name Martijn Gerretsen, wordt hartelijk bedankt voor de financiele

ondersteuning van het hele promotieonderzoek. Op een geheel vrijblijvende, maar altijd

geinteresseerde en behulpzame manier hebben zij het SENSE project vanaf de zijlijn begeleid.

Een persoon die zeker niet mag ontbreken in mijn dankwoord is Hans Kuster. Hans, jij hebt

een onmiskenbaar vermogen om andere mensen te enthousiasmeren en ik kan je vertellen dat het

effect niet alleen in Rotterdam waarneembaar is maar zelfs tot in Hoogeveen te voelen is.

155

Uiteraard moet ik eigenlijk mijn dank betuigen aan vrijwel de hele Amsterdamse en inmiddels

Rotterdamse Helicobacter pylori groep. Leo Smeets, het was aangenaam om samen met jou een

artikel te schrijven. Monique Gerrits en Raymond Pot, bedankt voor het verrichten van

ververschillende laboratorium bepalingen. Verder wil ik de vaste congres club (Arnoud van Vliet,

Ramon de Jonge, Nicolette de Vries en Jetta Bijlsma) bedanken voor de interessante discussies op

aangename locaties. Hopelijk kunnen we deze traditie in iedere geval voortzetten op de

voorjaarsvergaderingen van de NVMM.

Leen-Jan van Doorn wil ik bedanken voor de zeer plezierige en constructieve samenwerking

tijdens het schrijven van mijn eerste artikel. Zonder jouw relevante commentaren was het zeker

niet gelukt het artikel in zo'n goed blad gepubliceerd te krijgen. Wim Sluiter wil ik bedanken voor

de solide statistische analyse van het SENSE artikel. De beoordelingscommissie : prof. dr. E.J.

Kuipers, prof. dr. H.A. Verbrugh en prof. dr. J.B.M.J. Jansen wil ik bedanken voor hun bereidheid

mijn proefschrift te beoordelen.

Naast mensen die direkt betrokken zijn bij je dagelijkse werkzaamheden zijn andere mensen

minstens even belangrijk om je op af te reageren en “je batterij weer op te laden”. Constantijn,

Anneke, Robert, Evelien, Oscar, Annelies en Martin enorm bedankt voor jullie ondersteuning,

motivering en interesse gedurende al die jaren.

Tot slot wil ik mijn familie bedanken, mijn ouders, Marike, Wynand, Roel en Antoinette.

Door jullie wist ik mij steeds gesteund en jullie gaven mij goede raad als ik daarom vroeg.

Als allerlaatste, maar wel als meest belangrijke wil ik Charlie Kraan bedanken. Jij bent de

afgelopen jaren mijn steun en toeverlaat geweest in goede en slechte tijden en hopelijk wil jij dat

nog lange tijd blijven.

Bedankt !

Niek Arents

Arts-assistent Medische Microbiologie

Academisch Ziekenhuis Groningen

Postbus 30001

9700 RB Groningen

e-mail : [email protected]

Documents

University of Groningen The significance of Helicobacter ... · The significance of Helicobacter pylori in the approach of dyspepsia in primary care Contents Chapter 1 9 Introduction