Diagnosis of Helicobacter pylori: Invasive and non-invasive tests

Best Practice & Research Clinical GastroenterologyVol. 21, No. 2, pp. 299e313, 2007

doi:10.1016/j.bpg.2006.11.002available online at http://www.sciencedirect.com

6

Diagnosis of Helicobacter pylori: Invasive

and non-invasive tests

Chiara RicciLecturer of Internal Medicine

Gastroenterology Unit, University of Brescia, Italy

John HoltonSenior Lecturer in Microbiology

Centre for Infectious Diseases and International Health, Royal Free and University

College London Medical School, UK

Dino Vaira*

Associate Professor of Internal Medicine

Department of Internal Medicine and Gastroenterology, Nuove Patologie, University of Bologna,

Via Massarenti 9, 40138 Bologna, Italy

Helicobacter pylori infection can be diagnosed by invasive techniques requiring endoscopy and biopsy(e.g. histological examination, culture and rapid urease test) and by non-invasive techniques, such asserology, the urea breath test, urine/blood or detection of H. pylori antigen in stool specimen. Somenon-invasive tests, such as the urea breath test and the stool antigen test, detect active infection:these are called ‘active tests’. Non-invasive tests (e.g. serology, urine, near-patient tests) are markersof exposure to H. pylori but do not indicate if active infection is ongoing; these are ‘passive tests’.Non-invasive test-and-treat strategies are widely recommended in the primary care setting. Thechoice of appropriate test depends on the pre-test probability of infection, the characteristics ofthe test being used and its cost-effectiveness.

Key words: antigen test; Helicobacter pylori; invasive tests; non-invasive tests; stool; urea breathtest.

* Corresponding author. Tel.: þ39 051 636 4140; Fax: þ39 051 398 794.

E-mail address: [email protected] (D. Vaira).

1521-6918/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved.

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300 C. Ricci et al

INTRODUCTION

The incidental discovery, in 1983, of a gastric bacterium led to a dramatic change in thefield of gastroenterology.1 Helicobacter pylori infects more than half the global popula-tion, causing peptic ulcer disease and chronic gastritis; it is also strongly associatedwith gastric malignancies. Indeed, it has been classified as a class I carcinogen.2 H. pyloriinfection can be diagnosed by invasive techniques (endoscopy with biopsies for histol-ogy, culture and a rapid urease test) and non-invasive techniques (e.g. serology, the13C-urea breath test and the stool antigen test).3

All the available tests seem to be accurate in the diagnosis of H. pylori. However,a single test (with the exception of culture) is not sufficient to make a diagnosis ofinfection. For this reason, the European Guidelines take the gold standard to begenerally represented by at least two different tests.4 Despite this, it is usual in dailyclinical practice to use just one test for diagnosis of infection; choosing the ‘righttest’ is therefore most important.

Any diagnostic test can be characterised by its sensitivity and specificity, and by itspositive and negative predictive values; the latter parameters vary with the prevalenceof the disease in the community. The prevalence of a condition is the proportion ofindividuals in a population with the condition. A perfect test will correctly identifyall those who have the disease by recording a positive test (sensitivity 100%). Numer-ically, this is the number of true positive (TP) divided by the number of TP plus thefalse negative (FN): TP/(TP þ FN). A perfect test will also identify all those who donot have the disease by recording a true negative (TN) test (specificity 100%): TN/(TN þ FP). For example, if the prevalence of a condition is 40% (i.e. 400/1000 havethe disease) and the test identifies 340/400 persons with the disease correctly, thetest sensitivity is 85%; if the test identifies 480/600 persons without the disease cor-rectly, the specificity is 80%. If the prevalence of disease was only 10% the sensitivityand specificity would be unchanged and would detect 85/100 as positive and 720/900as negative.

There is generally a compromise between sensitivity and specificity in the test char-acteristics. For diseases that are amenable to treatment, a test with a high sensitivity ispreferable, whereas for those conditions that are untreatable or have implications forthe population at large, a test with a high specificity is required.

However, although they are reported in the literature more often than not, sensi-tivity and specificity are not particularly helpful; an alternative way to define them is:the probability of persons with the disease having a positive test (sensitivity) and theprobability of persons without the disease having a negative test (specificity). What ismore relevant is the probability of a person having (or not having) the disease givena positive (or negative) test. Such a calculation is possible using Bayes’ equation:

Probability of disease with a positive test¼ ðprevalence� sensitivityÞ=ðprevalence� sensitivityÞ þ ½ð1� prevalenceÞ � ð1� specificityÞ�

The Bayesian approach requires a pretest probability (in this case prevalence) to beconverted into a post-test probability. In Bayes’ theorem, the post-test probability isproportional to the pretest probability multiplied by the likelihood of the test beingpositive. This value is the positive predictive value (PPV) related to the prevalenceof disease in the community. Thus, in populations with a low prevalence of disease,the PPV will be low even if the sensitivity and specificity are high, and there will be

Diagnosis of Helicobacter pylori 301

many false-positive results. The converse is true of the negative predictive value(NPV):

NPV¼ Specificity� ð1� prevalenceÞ=½ð1� sensitivityÞ � prevalence�þ ½specificity� ð1� prevalenceÞ�

The likelihood ratio (the ratio of the probability of a positive result in a person withthe disease compared with the probability of positive results in a person who does nothave the disease) can indicate the value of test.

The choice of test should therefore be based on: (1) the prevalence of the infectionin the population; (2) the symptoms, such as the presence of alarm symptoms; (3) thelikelihood ratio for a positive and negative test; (4) the costs; and (5) the availability ofthe tests in the different settings.

Endoscopy was commonly used in diagnostic units but this procedure increasescosts and waiting times. More recently, the ‘test-and-treat’ strategy has been suggested.This uses a non-invasive test in patients without alarm symptoms (anaemia, weight loss,etc.), who are aged <55 years and who are not taking non-steroidal anti-inflammatorydrugs (NSAIDs). Endoscopy is still necessary when the patient is>55 years, if there arealarm symptoms or if there are acute new dyspeptic symptoms.5

INVASIVE TESTS

Endoscopy

Upper gastrointestinal endoscopy is an expensive and unpleasant procedure that car-ries the risk of haemorrhage and perforation; it has a reported mortality of 0.008% anda morbidity of 0.432%.6 The European Helicobacter Study Group (EHSG) Maastricht-2Report recommended that, in the primary care environment, patients who were<45e50 years of age, were presenting with dyspepsia in the absence of alarm symp-toms or symptoms of gastro-oesophageal reflux disease (heartburn, regurgitation) andwho were not taking NSAIDs, should be offered diagnosis by a test-and-treat strategyusing the non-invasive urea breath test or the faecal antigen test. A search-and-treatstrategy was recommended for patients with peptic ulcer who were on long-termacid-suppressive treatment. Endoscopy should be performed in patients <45e50years who had alarm symptoms and in all patients over this age limit, irrespective ofalarm symptoms. This strategy was endorsed by the EHSG Maastricht-3 guidelinesand supported by a number of publications, including a Cochrane systematic review.7e11

The most significant findings were a reduction in the number of endoscopies and a pricedifference of £160 for the test-and-treat compared with £400 for the endoscopy strat-egy. Endoscopies had a marginal advantage in older patients but even here there wasa definite cost saving. Further recommendations of the EHSG Maastricht-3 guidelineswere that a test-and-treat strategy was optimal in all adult patients with functionaldyspepsia in areas of high Helicobacter prevalence but that its efficacy was less in lowHelicobacter prevalence areas; an option here being empirical acid suppression.

In recent years there has been increasing interest in the goal of endoscopic in-vivohistology using narrow band imaging, chromoendoscopy and confocal laser endomi-croscopy, where the mucosal surface and subsurface can be examined in detail forthe presence of characteristic pathological features and the detection of H. pylori.12,13

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Narrow band imaging endoscopy is based on the principle of splitting white light intored, green and blue by narrow band filters (thus preventing wavelength overlap); thewavelengths being reflected from the mucosal surfaces at different depths. The inte-grated final image has the majority contribution from the blue part of the spectrumand reveals the surface microvasculature.

Confocal laser endomicroscopy was developed by including a confocal laser micro-scope in the tip of a video endoscope. The diameter of the insertion tube is 12.8 mm,indicating a high degree of miniaturisation. Images can be viewed at a resolution of1024 pixels with a field of view of 500 mm2 and at a depth of 250 mm. A contrast agent(either acriflavin or fluorescein) has to be used to see the cellular details. Fluoresceindoes not stain nuclei (given intravenously) whereas acriflavin (a topical agent) does andis thus of use in determining nuclear abnormalities and in combination with fluoresceinhas also been used to detect the presence of H. pylori on the mucosal surface.14 Theendoscopic appearance of the normal stomach is a regular arrangement of the epithe-lial cells, gastric pits and the subepithelial capillaries, which give a honeycombappearance. In H. pylori gastritis, the collecting venules cannot be seen, the gastricpits are enlarged with surrounding erythema and the normal capillary network islost (Figures 1 and 2). In atrophic gastritis, both the gastric pits and capillary networkare lost. In gastric cancer, there is loss of the capillary network, irregular branchedtubules and loss of polarity and pleomorphism of the nuclei.

Several conditions are characterised by prominent mucosal folds, which can be seenby endoscopy. In lymphocytic gastritis, which is thought to be an unusual reaction toH. pylori and is also found in coeliac disease, the surface mucosa can appear normal,although a typical appearance is varioliform, having prominent rugal folds carryingnodular elevations with white erosions at the apex and surrounded by a hyperaemicmargin.15 In Menetrier’s disease there are also prominent folds, which, on endosono-graphy, reveal hyperplasia of the deep mucosa (layer 2).16 The regularity of the epithe-lial cells, arrangement of the gastric pits and nuclear morphology can thus all be usedto make a real-time pathological diagnosis. Additionally, this advance in endoscopictechnique opens the way for the development of real-time in-vivo sensitivity testing.

Endoscopists should be specially trained in gastric histopathology. A shortcoming ofthese endomicroscopic techniques is an inability to view very far below the mucosalsurface. Endoscopy also provides biopsy specimens that can be used in different diag-nostic tests such as culture, histology, rapid urease test and the polymerase chainreaction (PCR).

Figure 1. Confocal laser endoscopy: H. pylori gastritis (from Ref. 14).


Histology

Histology can reveal the presence of bacteria as well as the type of inflammation. Manystains have been used to detect H. pylori, for example, WarthineStarry, Hp silver stain,Dieterle, Giemsa, Giminez, acridine orange, McMullen and immunostaining. Currently,the guidelines suggest that at least two stains are used: haematoxylin & eosin to eval-uate the inflammatory cells and the Giemsa or Genta stain to detect H. pylori. TheGenta stain has the advantage of visualising both the inflammatory cells and H. pyloriby combining a silver stain, haematoxylin & eosin and Alcian blue, although this is tech-nically complex and uses uranyl nitrate in its original formulation. Overall, the Giemsastain is the preferred stain for detecting H. pylori because of its technical simplicity, highsensitivity and low cost.17,18 Despite the high sensitivity of histology, the site, the num-bers and the size of the biopsies affect the diagnostic accuracy. Patchy colonisation canalso cause misdiagnosis. Everybody should be aware that although a single biopsy takenin the lesser curve, close to the angulus, can detect H. pylori presence in more than90%, the accuracy could be increased with multiple biopsies from the greater curveand corpus. Generally, specificity is high because of the peculiar morphology and itsclose relation to gastric mucosa.19

The histological appearance of gastritis uses the updated Sydney system. This hasa visual analogue scale with semi-quantitative scoring of mild, moderate and marked,and can score the density of H. pylori, granulocyte (acute gastritis) and mononuclearcell (chronic gastritis) infiltration, atrophy and intestinal metaplasia.20 There is a markedinfiltration of the lamina propria by granulocytes in acute gastritis; in chronic gastritisthis becomes a predominantly mononuclear infiltration.

In a histological section, H. pylori is recognised by its appearance as a short, curved orspiral bacillus resting on the epithelial surface or in the mucus layer; it is also found deep inthe gastric pits. Other Helicobacter species, such as H. heilmanii and H. bizzozeroni, are alsodetected in the human stomach. H. heilmanii is prevalent in about 0.1% of gastric biopsies.Its appearance differs from that of H. pylori: it is straight, rather than curved, and is muchlonger (about 10 mm), with tight spirals giving it a corkscrew shape. Gastritis associatedwith H. heilmanii has a characteristic histology of lymphocyte infiltration into gastric fo-veolae with lack of mucus depletion. H. heilmanii is a zoonotic infection in humans, beingderived from cats or dogs, and can give rise to chronic gastritis and possible MALT lym-phoma.21 H. bizzozeroni is similar to in appearance to H. heilmanii.

Figure 2. H. pylori gastritis (from Ref. 14).

304 C. Ricci et al

The sensitivity and specificity of histology for the diagnosis of H. pylori varies from53% to 90%, depending partly on the clinical setting, partly on the density of coloni-sation and partly on the experience of the histopathologist.

In general, a histological diagnosis can be made in about 90% of cases.22,23 Theaverage time for a histological diagnosis is 2e3 days, However, this increases whenmultiple biopsies are taken, which also increases the processing costs of the biopsiesand the overall costs of the diagnosis. Prior treatment to reduce the numbers ofH. pylori will adversely affect the sensitivity of histology.

In addition to the typical appearance of acute or chronic gastritis associated withH. pylori or non-pylori Helicobacter, other forms of gastritis that are associated withHelicobacter infection occur, such as lymphocytic gastritis and Menetrier’s gastritis.

Lymphocytic gastritis is characterised by the accumulation of small lymphocytes inthe surface and foveolae epithelium. A value of >25 intraepithelial lymphocytes/100epithelial cells is the diagnostic criterion. This is present in about 1% of unselectedendoscopies and in about 4% of patients with chronic gastritis. The histological appear-ance correlates well with the endomicroscopic appearance of varioliform gastritis.15

In Menetrier’s gastritis, which is thought to be associated with H. pylori, there is ahyperplastic epithelium with elongated, dilated and tortuous gastric glands penetratingthe muscularis mucosae, cyst formation and an oedematous lamina propria with an in-flammatory cell infiltrate when in the presence of H. pylori.24

MALT lymphomas, the majority of which are B cell in origin, have been defined asinfiltrates of centrocytoid cells, which have the appearance of plasma cells and whichdestroy the foveolae or gastric glands. The lymphomatous cells infiltrate all layersof the stomach. In low-grade lymphomas, the architecture is maintained and the celllineage spans the histological range from a small lymphocyte, to a centrocyte-likecell, to a monocyte-like B cell, to a lymphoplasma cell to a centroblast cell.25

Culture

Helicobacter can be cultured from gastric biopsies. The colonies are identified bya Gram stain and biochemical tests. The colonies are Gram negative, urease positive,oxidase positive and catalase positive. Helicobacter is very fragile out of the gastric en-vironment: this is why the culture has to be processed as soon as possible. The biop-sies can be kept in a transport medium (Stuart’s transport medium) for 24 h at 4 �C.Helicobacter are isolated on agar (Columbia or braineheart infusion), generally withadded antibiotics and albumin. The agar plates are incubated in a micro-aerobic envi-ronment, which is obtained by using a jar with a gas-generating kit for a micro-aerobicatmosphere (5% oxygen and 5e10% CO2). The plates are incubated for at least 5 daysat 37 �C, even though Helicobacter colonies sometimes appear after just 3 days.

Although culture has a high specificity (100%), the sensitivity is often lower. This mightbe because: an insufficient number of biopsies was taken, there was a delay in transport-ing the culture to the laboratory, the culture was exposed to an aerobic environment orthe cultures might not have been recognised as a result of microbiological inexperience.Moreover, antibiotics, proton pump inhibitors and H2 antagonists must not taken in thepreceding 2 weeks because they also reduce the sensitivity of culture.

Culture tends to be done only in research centres particularly dedicated to H. pyloriinfection. However, as the prevalence of antibiotic resistance increases globally there isa strong argument for performing culture and sensitivity testing after the first treat-ment failure (to prevent the emergence of double resistance to clarithromycin and


metronidazole), and certainly after the second. Indeed, some would argue that itshould be performed at the initial diagnosis in areas of high resistance prevalence.26e28

Also, it has to be emphasised that susceptibility for a full range of the antibiotics canonly be done on culture. When testing for antibiotic sensitivity on a routine basis, thetwo most used tests are disc diffusion and the E-test, with the agar dilution test as thereference. For clarithromycin and amoxycillin there is a good correlation between theE-test and agar dilution, however, for metronidazole the E-test tends to give higherMICs compared to the reference method or the disc test.29,30 An additional importantreason for culturing H. pylori is for research purposes, such as correlating specificvirulence characteristics with different clinical outcome and investigation of the molec-ular events following binding of the organism to gastric epithelial cells.

Rapid urease test

This gastric biopsy test is based on the activity of the H. pylori urease enzyme, whichsplits the urea test reagent to form ammonia. Ammonia increases pH, which is de-tected by the indicator phenol red. Although some of the commensal flora of the oro-pharynx produce urease, which is swallowed in the saliva, this weaker enzyme isdenaturated rapidly in the acid lumen of the stomach (pH < 2.0). Many commercialurease tests are available, including gel-based tests (CLOtest, HpFast) paper-basedtests (PyloriTek, ProntoDry HpOne) and liquid-based tests (CPtest, EndoscHp). Thetests give a result in 1 h to 24 h, depending in part on the format of the test andthe number of Helicobacter in the biopsy specimen. In some tests (PyloriTek), the sug-gested advantage is the built-in positive and negative control with each test strip, andthe fact results are obtained within an hour, as opposed to, for example, the CLOtest,which takes 24 h. Unbuffered tests can give results in less than 1 h, e.g. the CPtest, andthese liquid tests can be prepared cheaply in-house by adding phenol red to a solutionof urea and adjusting the pH to 6.6. At this pH the indicator is yellow and, on additionof a positive biopsy, will convert the solution to a red colour. One disadvantage of in-house tests, apart from quality control, is a short shelf life. All the commercial rapidurease tests have specificities above 95e100% but the sensitivity is slightly less, at85e95%.31e36 In comparison to histology, culture and PCR, urease tests are morerapid, much cheaper and have comparable sensitivity and specificity (except for cul-ture, which has 100% specificity).

The sensitivity is affected mainly by the number of bacteria present in the biopsy. Ithas been calculated that 104 organisms are required for a positive result and a propor-tion of patients can harbour densities lower than this. Low sensitivity and specificityare also reported in post-treatment and in bleeding patients, and for these reasonstheir use is not advised in these clinical settings.

False-negative urease test can also be obtained in patients with achlorhydria as wellas in patients on proton pump inhibitors because the increased luminal pH can lead toextremely high pH adjacent to the organism, such that H. pylori is destroyed by theaction of its own urease.37

Molecular tests

Two molecular tests that are available are in-situ hybridisation and the PCR. In-situhybridisation has been used to detect the presence of H. pylori in archival specimens

306 C. Ricci et al

and to detect the presence of specific virulence markers; however, it has more oftenbeen used to detect clarithromycin resistance.38e41 The sensitivity and specificity forthe diagnosis of H. pylori infection using in-situ hybridisation with biotinylated probeshas been reported at 95% and 100%.

PCR has been used extensively for the diagnosis of H. pylori from gastric biopsyspecimens, saliva, faeces and archival specimens, as well as for detecting clarithromycinresistance.42e44 PCR yields information on the presence of potential virulencemarkers in the strain, which might have implications for the development of severedisease or efficacy of eradication. Different primers have been utilised and somehave been developed into commercial kits. Different loci have been used as the targetfor the amplification: 16S rRNA; A-, B- and C-urease; flaA; cagA; vacA and heat-shockprotein (hsp). Real-time results can be obtained using light-cycle technology.

PCR detection from faeces is less generally used, partly because cheaper, technicallyeasier and more reliable tests are available and partly because some faeces are repletein inhibitors of the PCR reaction. PCR is also been used to test the chemosusceptibil-ity of Helicobacter to clarithromycin, which is used in the standard eradication regimen,because resistance to clarithromycin is caused by point mutations (A-G translocation)in the 23S rRNA gene.45 It is more difficult to detect multiple mutations, although it ispossible with a line probe assay (LiPA) using biotinylated probes for the different mu-tations or after amplification with a preferential homoduplex formation assay(PHFA).46,47

Although currently a research tool and not generally used in routine diagnosis,microarrays might be used diagnostically for H. pylori in the future. The disadvantagesof PCR as a routine test are that it is a technically demanding and expensive test com-pared to culture, histology and the rapid urease tests. It requires special laboratoryconditions with separate facilities for each stage of the technique and, as it is highlysensitive, it is subject to false-positive results by contamination. A positive result de-tected by any of the molecular techniques does not indicate current infection but willalso detect the DNA or dead organisms.48

NON-INVASIVE TESTS

These tests can either be either active or passive. Active tests detect the presence ofH. pylori, i.e. provide evidence of a current infection. The stool test detects the pres-ence of bacterial antigens of H. pylori in stool; these antigens disappear when H. pyloriinfection is cured. The urea breath test is another direct test based on the detection ofurease activity, which is demonstrated in individuals with active H. pylori infection. Pas-sive tests provide evidence of exposure to H. pylori at some time and do not indicatewhether the infection is currently active. These tests are based on the detection ofantibodies to H. pylori.

Passive tests

Serological tests

There are three main formats for these tests: the most common is the enzyme-linkedimmunosorbent assay (ELISA) test, which detects the totality of the immunoglobulin ina patients’ serum and can detect any of the immunoglobulin isotypes. If the antibodyresponse to specific antigens is important, the separate ELISA test has to be done


for each antigen. This test is usually done in the laboratory because it requires anELISA reader.

Latex agglutination tests require only minimal equipment and can detect all theimmunoglobulin isotypes. They are performed more rapidly than an ELISA test andare commonly used for near-patient tests.

The third type of test is based on Western blotting: the specific antigens are sepa-rated by gel electrophoresis, transferred to a filter-paper strip and reacted with thepatient’s serum sample. These immunoblots detect all the immunoglobulin isotypesbut have the advantage of detecting specific antigens in one assay. Commercial immu-noblots are used in near-patient testing as they require minimal apparatus and are rapid.

Since the appearance of the first serological tests for the diagnosis of H. pylori in-fection, they have become the most frequently used tests in routine practice becauseof their accuracy, low cost and availability. Serological tests are based on the detectionof specific anti-H. pylori IgG antibodies in a patient’s serum. Some tests also detect thepresence of IgA in the saliva or IgG in the urine. The advantages of serological test arethat they require no specialised equipment or technique and can be performed bymost hospital or clinic laboratories.

The sensitivity and specificity of the tests depends on the antigen used, the clinicalcontext, the gold standard used as a comparator and the prevalence of H. pylori inthe community. Overall, the sensitivity has been reported (mostly for the ELISA-basedformat) as ranging between 90% and 97% and the specificity as between 50%and 96%.49e53

The most important disadvantage of serological tests is that they do not distinguishbetween active infection and a previous exposure to H. pylori. Antibody levels can per-sist in the blood of individuals cured of H. pylori infection for long periods of time.Therefore, as the numbers of patients successfully treated for H. pylori increase ina population, the prevalence of false-positive tests with serology increases. Recently,McNulty54 correctly stated that a positive serological test can mean: (1) that thepatient is infected at the time of the test; (2) that the patient was once infectedbut, by the time of the test, infection has resolved; or (3) that the test is detectingnon-specific cross-reacting antibodies. Therefore, using a serology-based test, 255of 2000 patients tested are likely to receive an incorrect diagnosis of active H. pyloriinfection and receive inappropriate treatment.54

CagA and vacA antibodies

CagA is a 120-kDa protein of H. pylori with high immunogenicity. Its gene (cagA) is con-tained in the pathogenicity island (PAI) of the chromosome of H. pylori. Individuals in-fected with cagA-positive strains of H. pylori tend to have more severe gastritis,a higher likelihood of developing gastric atrophy and intestinal metaplasia, and a higherincidence of duodenal ulcer and intestinal-type gastric cancer.12 The vacuolating toxin(vacA) is an 87-kDa protein and its gene is not located in the pathogenicity island.However, it is secreted by Helicobacter strains that contain the PAI. The vacuolatingtoxin (vacA) is activated at low pH, is resistant to acid and pepsin, and causes vacuo-lation of gastric cells in cell culture in vitro; in animal models it has been shown to dam-age mouse gastric epithelium.

Antibody tests still play an important role in studies of pathogenesis and virulence.Although these tests are of limited value in the clinical setting, they have increased ourunderstanding of the pathogenesis of disease caused by H. pylori. Antibodies against theimportant proteins of H. pylori, cagA and vacA, can be detected using different

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immunological techniques. After eradication of H. pylori, the antibodies disappear atdifferent times and some, such as the anti-CagA antibodies, may persist for years. Al-though the antibody response to cagA can be detected using an ELISA technique, im-munoblots have a particular advantage in that they are more sensitive and, asmentioned, can provide evidence of immunological reactions to several antigens allat once, which might have clinical relevance.49,55

Near-patient tests

Near-patient tests were developed to provide a rapid diagnosis of H. pylori infection inclinics or physician offices; they are also called office-based serology tests. They aretechnically simple to perform and the most convenient of them uses a drop of wholeblood obtained by finger-prick. Other near-patient tests use serum, require venepunc-ture and use a centrifuge to separate the serum. The results of finger-prick tests canvary significantly when the flow of blood is poor and there is difficulty in obtaininga drop of blood; squeezing the finger can express tissue fluid into the blood sample,thereby changing the concentration of antibody sample being studied. Whole-bloodtests are affected by circulating chylomicrons, which change the permeability of theblood sample to the various antigens in the membranes used in diagnostic tests. Stud-ies have reported a lower sensitivity and specificity than was originally suggested; themean sensitivity was of 71% and specificity of 87%. A Canadian study of serology foundthat 33% of positive near-patient tests in dyspeptic patients in primary care settingwere false positives.56 The current near-patient tests are not recommended for diag-nosis as they are likely to be false-positive results if the prevalence of the disease islow, even though the sensitivity may be high.

Tests on saliva and urine

Tests on saliva and urine are attractive because samples can be easily obtained. Theyshare the same probability as other antibody tests in the serum and have an additionalproblem in that the concentration of the antibody is lower than in serum, making de-tection more difficult. Individual centres have reported promising results (sensitivity81%, specificity 95%). However, these data were not confirmed in a multicentre trialusing the same urine assay kit, when a sensitivity of 89% and a specificity of 69% werereported.57

Active tests for the detection H. pylori

These tests are useful for the initial detection of H. pylori and for confirmingeradication.

13C-urea breath test

H. pylori produce urease, an enzyme that splits urea into ammonia and carbon dioxide.Indeed, the organism is thought to use urease activity to regulate pH in its micro-environment. The urea breath test is based on the principle that urease activity is pres-ent in the stomach of individuals infected with H. pylori. Patients ingest urea labelledwith either 13C or 14C. Hydrolysis of urea occurs within the mucus layer and resultsin the production of labelled CO2. The CO2 diffuses into the epithelial blood vesselsand, within a few minutes, the isotopic CO2 appears in the subject’s breath. Labelled


urea is usually given to the patient with a test meal to delay gastric emptying and in-crease contact time with the mucosa. After ingestion of the urea, breath samples arecollected for up to 20 min by exhaling into a CO2-trapping agent (hyamine). Ureabreath test have a very high sensitivity and specificity, ranging from 95% to 97%. How-ever, the urea breath test might not be reliable when assessing patients who have hadgastric surgery or those who have been on a proton pump inhibitor or ranitidine.58

The 13C urea breath test is similar to the 14C urea breath test except that 13C isa non-radioactive isotope of 12C and its detection requires a mass spectrometerrather than a scintillation counter. An alternative detection method is infra-red spec-troscopy, which is technically simpler and also cheaper than a mass spectrometer.59

Because 13C is a naturally occurring stable isotope, there are no nuclear regulatoryconcerns and the test can be used in children and pregnant woman. Since the initialreport of this test, it has been extensively modified, including variations in dose-labelled urea, sampling time, test meal and cut-off value. The standard urea breathtest uses 75 mg of 13C and was compared to a rapid low-dose (50 mg 13C) test for-mulated as a tablet also containing citric acid. The test was utilised both as a diagnosticand to evaluate clearance after eradication of H. pylori. It gave the standard test sen-sitivity and specificity of 100%. Doses of 13C as low as 10 or 15 mg also gave sensitivityand specificity of 89e96% and 100%, respectively, and the 15-mg dose was accuratefor assessing eradication.60,61

Stool antigen test

An enzymatic immunoassay (EIA), which detects the presence of H. pylori antigen instool specimen, has recently become available. This assay has undergone extensivetesting for the initial diagnosis of H. pylori infection and for confirmation of erad-ication after treatment. The most widely used test in the assay uses polyclonal anti-H. pylori-capture antibodies absorbed to microwells. This polyclonal antibody testhas been extensively evaluated in the diagnosis of H. pylori infection before therapy.Most studies have suggested that the test’s accuracy is similar to that of the ureabreath test in the initial diagnosis of H. pylori infection.62,63 A recent study sug-gested that the urea breath test and stool antigen test are equally accurate in con-firming eradication. A positive stool test 7 days after completion of treatment ispredictive of failed eradication.64

Very recently, Gisbert et al, have reported in a metanalysis paper, the superiority ofmonoclonal stool antigen compared to polyclonal test, both for the initial diagnosis ofH. pylori and for confirmation of its eradication after treatment. (Table 1).65 Accordingto the European Guidelines, the monoclonal test and the urea breath test are the twonon-invasive tests recommended for monitoring the success or failure of eradicationtreatment.5 A new rapid monoclonal one-step lateral flow immunoassay stool test,

Table 1. Monoclonal test overall results.

Pre-treatment

N ¼ 2499 patients

Post-treatment

N ¼ 957 patients

Sensitivity 94% 93%

Specificity 97% 96%

Source: Gisbert et al, Am J Gastroenterol 2006;101:1921e30.

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taking only 5 min, has been recently been released (Figure 3). Stool is placed in a diluentvial and then a drop of the liquid is placed in the well of the test. Early results arepromising, with sensitivity and a specificity of 92%.66 This test might be useful asa near-patient test in primary care settings. It also has a particular advantage in nee-dle-phobics and children, who are generally averse to venepuncture. In principle, thetest can be amended to pick-out specific proteins, such as cagA or vacA, which couldbe used to determine the virulence type of the infecting strain.

COST-EFFECTIVENESS AND STRATEGIES BASED ON PRETESTPROBABILITY OF INFECTION

The choice of an initial test for H. pylori detection depends on the prevalence of H. pyloriinfection and the value placed on increased diagnostic accuracy. A recent cost-benefitanalysis evaluated single test and the sequential testing strategy using costs in the UShealthcare system.67 The serology test had the lowest cost per correct diagnosis atlow (30%), intermediate (60%) and high (90%) prevalence ($90e95/correct diagnosis)but its diagnostic accuracy was low (80e84%). At low and intermediate prevalence, thestool test was more accurate (93%), with an average cost of $126e127 per correctdiagnosis and an incremental cost of $336e381 per additional correct diagnosis. ELISAtesting was preferable when prevalence rates were very high (90%) and using a confir-matory urea breath test when the ELISA test was negative increased the diagnosticaccuracy to 96% with modest incremental costs. If the cost of the breath test wasless than $50, or if the cost of the stool test was greater than $82, breath testingbecame preferable to stool testing. In patients with a<60% pretest probability of infec-tion, as is the case in dyspeptic patients in much of Western world, the stool testprovides increased accuracy with modest incremental costs.

Figure 3. ImmunoCard Stat! HpSA (from Ref. 66).


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Diagnosis of Helicobacter pylori: Invasive and non-invasive tests