Editorial
Vol. 11. No. 7
577
•Eur. J. Clin. Microbiol. Infect. Dis., July 1992, p. 577-582 0934-9723/92/07 0577-06 $ 3.00/0
An Assessment of Serological Tests for Detection of
Helicobacter pylori H. y o n Wulffen
Serological detection of Helicobacter pylori has been possible ever since the organism was first SUccessfully cultured by Marshall et al. in 1983 (1). Various methods have been employed including complement fixation (2, 3), agglutination (2), passive hemagglutination (4), indirect immunofluorescence (5), enzyme immunoassays (EIAs) (6-8) and immunoblot techniques (9, 10). All of these studies have shown that the vast majority of patients infected with Helicobacter pylori mount an easily detectable humoral immune response against this organism. Studies comparing the performance of older and more recent methods, for instance complement fixation compared with imrnunoblot (3), usually reveal superiority of the EIA and immunoblot methods over the other techniques. In the meantime a number of commercial tests, mostly based on the EIA technique, have appeared on the market. Two of these test kits have been evaluated in one of the three serological studies of Helicobacter pylori published in this issue of the journal (11). It can thus be expected that serological tests for Helicobacter Pylori will no longer be restricted to research purposes but will be included in the routine diagnostic work-up in patients with presumed gastrodUodenal pathology. For this reason it seems pertinent to reassess the current status of serological tests for Helicobacterpylori.
Immune Response Rathbone et al. (12) have shown that the immune response to Helicobacter pylori at the mucosal level is predominantly of the IgA type which is as could be expected in a chronic mucosal infection. Circulating antibodies against Helicobacter Pylori, however, are predominantly of the IgG class (12). IgG subclass analysis shows involvement of all four subclasses with a predominance Inslitut far Medizinisehe Mikrobiologie und Immunologie, Universitfitskrankenhaus Eppendorf, Martinistr, 52, 2000 Hamburg 20, Germany.
of the IgG1 and IgG2 subclasses (13). In this study of Bontkes et al. (13) patients with duodenal ulcers had a significantly higher IgG2 response than patients with chronic active gastritis without a duodenal ulcer. A systemic immune response of the IgA type usually seems to be less pronounced but if marked appears to indicate more severe inflammation (8, 14). For unknown reasons a small percentage of Helicobacter pylori-infected patients fail to mount a systemic IgG response and demonstrate IgA antibodies only. IgM antibodies are rarely found and seem to be of minor importance, however at present little information is available on acute Helicobacterpylori infection and subsequent seroconversion (15, 16).
Serological Techniques As mentioned above the full spectrum of serological techniques has been applied to Helicobacter pylori, the E I A and immunoblot methods having received the most attention. The immunoblot method has a very high sensitivity, one reason for this being the fact that any particular immunogenic protein concentrates in a very thin line on the nitrocellulose membrane. By using class-specific secondary antibodies the immune response can be differentiated with regard to the different immunoglobulin classes. As discussed below, it is frequently not so much the test method but the antigen preparation that determines the quality of a serological assay. The immunoblot technique is the best means for studying antigen preparations and their variations. For instance, comparative analysis of the protein profiles of different Helicobacter pylori isolates may help decide whether to use one or more strains as antigen. Immunoblot analysis may also reveal the presence or absence of crossreacting proteins in different preparations of the antigen. Thus, no matter what method is used for serological tests, the immunoblot is an excellent technique for quality control of an antigen
578
preparation. However, problems may also arise when using the immunoblot method as reference for other techniques. In complex mixtures such as a whole cell lysate or a sonicate where a very dense set of reactive bands results, it may be difficult to identify a particular protein band. Furthermore, depending on the quality and proportion of polyacrylamide and the method of transfer used, not all proteins of interest may be transferred to the nitrocellulose during the blotting procedure. Since the antigen samples are usually boiled in sodium dodecyl sulfate (SDS) and other denaturing agents, some of the immunodominant epitopes may no longer be recognized. Apart from that, the immunoblot technique is quite labor-intensive and thus not suitable for testing large numbers of sera. Moreover, it only allows semiquantitative interpretation of results. The EIA, on the other hand, is easy to perform and therefore well suited for screening purposes. It possesses comparable sensitivity, may also be used for detection of class-specific antibodies, and - a major advantage - results may be expressed quantitatively It is not necessary to use end-point titrations for this purpose. Results may be expressed as EIA units (U/ml) based on serial dilutions of an internal standard serum (8). Alternatively, the concentration of anti-Helicobacter pylori antibodies can be measured by using a standard curve for human immunoglobulin (17), the results being expressed as the amount in micrograms of bound immunoglobulin per millilitre. An interesting variation of the EIA is the transferable solid phase EIA technique (TSP-EIA) or pin-plate technique employed by Faulde et al. (18) which is described in this issue of the journal. It allows synchronisation of processing during all incubation steps thereby eliminating intra-assay variations caused by time delays in processing between the first and last wells of a plate.
Choice of Antigen Early studies employed crude antigens such as whole cells or whole cell sonicates with acceptable results (2, 6, 12). Improved performance values were reported using partially purified antigens such as acid glycine extracts (7, 8, 19) or an ultracentrifuged supernatant from a whole cell sonicate (20). More recently, attempts have been made to use purified antigens to further reduce cross-reactivity and improve specificity. When
Eur. J. Clin. Microbiol. Infect. Dis.
highly purified antigens such as the Helicobacter pylori urease (21) or the 120 kDa protein, as described by Gerstenecker et al. (22) in their paper in this issue of the journal, have been employed, specificity was indeed increased. However, not all Helicobacterpylori-infected patients demonstrated antibodies against these antigens, and thus the sensitivity was lower. For the 120 kDa protein this can be partly explained by the fact that not all infecting Helicobacter pylori strains exhibit this particular protein (23). It has therefore been suggested that two or more purified antigens be combined in one EIA. Evans et al. (24) describe an assay which only employs high molecular weight cell-associated proteins (HM-CAP) and which apparently has both high sensitivity and specificity. Hirschl et al. (25) reported similar results using high molecular weight proteins between 80 and 130 kDa. One of the commercially available test kits (Cobas Core Anti-Helicobacter pylori EIA, Hoffmann LaRoche, Switzerland) combines a number of fast protein liquid chromatography (FPLC) fractions including the native urease enzyme and performs favorably according to Goossens et al. (26). Other manufacturers give very little information on their antigen preparations, but certainly some of them use partially purified antigens such as acidglycine extracts. The importance of using highly purified antigens is still a matter of controversy. The main crossreactivity reported is between the flagellar proteins of Helicobacter pylori and Campylobacterjejuni in the 54-59 kDa region (27, 28). However, in a recent immunoblot study conducted by Andersen et al. (29), serological cross-reactions with other bacteria were very limited. In particular, in absorption studies only one minor reactive band of approximately 100 kDa was absorbed from a Helicobacterpylori-positive serum pool by Campylobacterjejuni and Campylobacter coli strains. Talley et al. (14) compared four antigen preparations, namely a sonicate, an acid-extractable surface antigen, an acid-extractable antigen from an aflagellate variant (to reduce cross-reactivity with Campylobacterjejuni), and a FPLC-fractionated urease-containing antigen. They found that the diagnostic value of the different EIAs was highly comparable, and that the crude antigens performed as well as the more purified antigens, the FPLC-purified antigen displaying the lowest sensitivity. However, crude antigens usually give higher backgrounds which may make it difficult to establish optimal cut-off values to discriminate between low positive and
Vo1._._211,1992
true negative results. Therefore some sort of purified multi-component antigen is certainly desirable to achieve both high sensitivity and specificity. The question arises whether the antigen preparation should be derived from one or more strains of Helicobacter pylori. Several studies (27, 29-3I) have shown that there are only minor differences in antigenic composition between different isolates of Helicobacter pylori. Nevertheless, it has also been reported that sera from some Helicobacter pylori-infected patients can have a negative reaction with antigen from one strain but a Positive reaction with antigen from other strains (32).
Evaluation of Serological Tests
579
under study as impressively demonstrated in the recent study of Goossens et al. (26). These authors evaluated the Cobas Core AntiHelicobacter pylori E I A (Hoffmann-LaRoche, Switzerland) in 855 Belgian and 257 Mediterranean patients in whom upper gastrointestinal endoscopy was clinically indicated. They obtained a very good negative predictive value of 96 % in the Belgian population where there was an age-dependent prevalence of Helicobacter pylori infection ranging from 34 to 53 %. In the Mediterranean group, on the other hand, where there was a high prevalence of Helicobacterpylori infection of 87 %, they achieved a rather poor negative predictive value of 64 %. Specificity of the assay also dropped from 95 % in the Belgian group to a modest 70 % in the Mediterranean group.
Serological assays are usually evaluated on the basis of performance criteria such as sensitivity, Specificity, and positive and negative predictive Values. To obtain these data, a set of clearly defined positive and negative reference sera is needed. This raises the issue of the reference method. For the purist among the microbiologists culture of the organism should serve as reference. However, the distribution of Helicobacter pylori and the related type B gastritis in the stomach rnucosa is irregular so that the question arises as to how many biopsies and cultures are needed to obtain 100 % for the proposed reference method. Depending on the quality of the reference method, a serological technique may appear to perform inferiorly even if in fact it is more acCUrate. Whether a biopsy urease test, urea breath tests, microscopic detection of the typical organism or even the histopathologist's diagnosis of type B gastritis come into question as reference method for serological tests probably depends on the quality of the reference methods available in a given situation. Using the same acid glycine extract as antigen for an IgG EIA, our team arrived at quite different performance values depending on the reference method employed. Overall, we obtained the best values using a 14C-urea breath test as reference (33).
As the performance values relate to the chosen cut-off value and the prevalence of the infection in the patient population, it may be advisable to re-evaluate a test before use if it has originally been tested in a different patient population. This also holds true for commercial test kits, as has been clearly demonstrated by van den Oever et al. (34). When these authors evaluated the commercial GAP test (Bio-Rad Laboratories, USA) following the manufacturer's instructions, they achieved values for sensitivity, specificity, the positive predictive value and negative predictive value of 32.7 %, 100 %, 100 % and 58.9 % respectively. They then established a receiver operating characteristic (ROC) curve which involves plotting true-positive rates against false-positive rates for various possible cut-off values. The cut-off value whose R O C curve encompasses the largest area is considered to be the most accurate. By establishing the optimal cut-off value of the G.A.R test for their population in this manner, they arrived at values for sensitivity, specificity, the positive predictive value and negative predictive value of 87.8%, 91.3 %, 95.6 % and 77.8% respectively, which seems more feasible. Another example of use of such R O C curves for determination of the optimal cut-off value and for comparing the quality of different assays is given in the study of Aguirre et al. (11) presented in this issue of the journal.
Care is necessary in evaluating different EIAs solely on the basis of performance values when these values have been derived from different Populations. The negative predictive value, in particular, is highly dependent on the actual prevalence of the infection in the population
While possible cross-reactions may not be relevant in one population, they may very well be of importance in a different population. This has been shown by Mitchell et al. (35) who employed the same sonicated antigen in an EIA for screening both a Sydney population and sera from a
580
Papua New Guinean population. While absorption of sera with Campylobacter jejuni had little effect on the results in the Sydney patients, this proved to be a crucial step in the Papua New Guinean population. Without the absorption step the Helicobacter pylori infection rate in Papua New Guineans appeared to be 56 %, whereas use of the absorption step reduced this rate to 25 %. Thus, in populations with a high prevalence of anti-Campylobacter jejuni antibodies these findrags should be taken into account.
Applications of Serological Tests Serological tests provide a practical and non-invasive means of assessing Helicobacter pylori infection in an individual patient or in a large population. The only non-invasive alternative is urea breath tests, however these require special equipment, time for collection of breath samples, and use of either radioactive material or tracers that can only be measured with mass spectrometers. • One of the main fields of application of serological tests to detect Helicobacterpylori is epidemiology. In gastric cancer epidemiology such tests have become an indispensable tool, the best evidence we have today of an association between chronic Heticobacter pylori infection and gastric cancer being obtained in serologic screening of high-risk populations (36, 37). Another important indication for such serological investigations is in the long-term follow-up of Helicobacter pylori eradication therapy. Several authors could show that significant falls in antibody titers usually occur within about six months after successful eradication therapy (8, 16, 38). Again, IgG titers give the best results. Peptic ulcer disease is not a rare finding in childhood, and abdominal pain even less so. Use of upper gastrointestinal endoscopy, however, is usually avoided or delayed in these patients as, among other things, it can involve general anaesthesia. Serological tests for Helicobacter pylori might be very helpful in this patient population for screening. Again, this is a population where the mode of evaluation may have to be changed. Thus it has been suggested that cut-off values may have to be adjusted in pediatric patients (39-41). Suggestions have also been made to employ serological tests for Helicobacter pylori in adult patients to lower the endoscopy load and thereby
Eur. J. Clin. Microbiol. Infect. Dis.
costs. By using a screening strategy based on age, serological tests for Helicobacter pylori and documentation of use of non-steroidal anti-inflammatory drugs (NSAIDs), Sobala et al. (42) demonstrated that they might have reduced the endoscopy workload by 23.3 % and would have had a sensitivity in the detection of peptic ulcers of 97.4 %. No case of malignant disease would have been overlooked although 6 of 192 cases of peptic ulcer in a group of 842 patients would not have been detected. A policy of symptom-based screening in dyspeptic patients under the age of 45 would in fact have been less sensitive than the proposed screening based on serological tests for Helicobacter pylori whereby only those patients underwent endoscopy who were either sero-positive for Helicobacter pylori, taking NSAIDs or aged 45 years or more. The age limit of 45 was chosen to minimise the risk of overlooking cases of upper gastrointestinal malignancy. Serological tests for Heticobacter pylori might also be effectively employed for screening certain high-risk patients as we demonstrated in a retrospective study of patients before and after renal transplantation (43). Such patients may have been infected with Helicobacter pylori for many years without overt disease until an event such as a transplantation compromises their immune system, including the mucosal defense mechanisms of the stomach and duodenum. Early serodiagnosis and appropriate therapy aimed at eradication of Helicobacter pylori might possibly prevent these patients from developing peptic ulcers and gastrointestinal hemorrhage.
Conclusions Serological tests provide an easy and non-invasive means of detecting Helicobacter pylori infection. Testing for IgG antibody usually suffices; testing for lgA may be complementary in some instances. A number of commercial assays are now available on the market, and such tests will undoubtedly become part of the routine diagnostic work-up. However, the performance values (sensitivity, specificity, predictive values) reported for these tests have rarely been above 90 % which is not very satisfactory (11, 26, 34, 4446). Furthermore, the optimal antigen composition has not yet been determined. Cut-off values proposed by the manufacturers are often not satisfactory and should be re-evaluated for a particular population. It must also be taken into ac-
V ol. 11,1992
count that performance values are closely interrelated to the prevalence of the infection in the population under study. Serological tests for Helicobacter pylori are particularly useful for monitoring the results of eradication therapy. They may also be useful for screening certain patient populations to help decide whether to perform endoscopy provided certain rules are strictly adhered to. It is not advisable, however, to base the diagnosis and treatment of patients solely on positive serological tests for Helicobacter pylori, a practice which is unfortunately already gaining ground.
References 1. Marshall B J, Royce H, Annear D J, Goodwin CS, Pearman JW, Warren JR, Armstrong JA: Original isolation of Campylobacter pyloridis from human gastric mucosa. Microbios 1984, 25: 83--88. 2. Jones DM, Lessels AM, Eldridge J: Campylobacter like organisms on the gastric mucosa: culture, histological and serological studies. Journal of Clinical Pathology 1984, 37: 1002-1006. 3. yon Wuiffen H, Heesemann J, Biitzow GH, LOning T, Lanfs R: Detection of Campylobacter pyloridis in patients with antrum gastritis and peptic ulcers by culture, complement fixation test, and immunoblot. Journal of Clinical Microbiology 1986, 24: 716-720. 4. Marshall BJ, McGechie DB, Francis G J, Ulley PG: Pyloric campylobacter serology. Lancet 1984, it: 281, 5. Faulde M, Putzker M, Metres T, Sobe D: Evaluation of an immunofluorescence assay for specific detection of immunoglobulin G antibodies directed against Helicobacter pylori, and antigenic cross-reactivity between H. pylori and Campylobacter jejuni. Journal of Clinical Microbiology 1991, 29: 323-327. 6. Booth L, Holdstock G, MacBride H, Hawlin P, Gibson JR, Ireland A, Bamforth J, DuBoulay CE, Lloyd RS, Pearson A: Clinical importance of Campylobacter pyloridis and associated serum IgG and IgA antibody responses in patients undergoing upper gastrointestinal endoscopy. Journal of Clinical Pathology 1986, 39: 215-219. 7. Goodwin CS, Blincow E, Peterson G, Sanderson C, Cheng W, Marshall B J, Warren JR, McCulloch R: Enzyme-linked immunosorbent assay for Campylobacter pyloridis: correlation with presence of C. pyloridis in the gastric mucosa. Journal of Infectious Diseases 1987, 155: 488-494. 8. yon Wulffen H, Grote H J: Enzyme-linked immunosorbent assay for detection of immunoglobulin A and G-antibodies to Campytobacterpylori. European Journal of Clinical Microbiology & Infectious Diseases 1988, 7: 559-565. 9. Kaldor J, Tee W, Nicolacopolous L, Demirlzoglou K, Noonan D, Dwyer B: Immunoblot confirmation of immune response to Campylobacter pyloridis in patients with duodenal ulcers. Medical Journal of Australia 1986, 145: 133-135.
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10. von Wulffen H, Grote H J, Gatermann S, Liining T, Berger B, Buhl C: Immunoblot analysis of immune response to Campylobacter pylori and its clinical associations. Journal of Clinical Pathology 1988, 41: 653659. I1. Aguirre PM, Pascual CY, Merino FJ, Velasco AC: Evaluation of two commercial enzyme immunoassays for the diagnosis of Helicobacter pylori infection. European Journal of Clinical Microbiology and Infectious Diseases 1992, 11: 634--639. 12. Ralhbone BJ, Wyatt Jl, Worsley BW, Shires SE, Trejdosiewicz LK, Heatley RV, Losowsky MS: Systemic and local antibody responses to Campylobacter pyloridis in non-ulcer dyspepsia. Gut 1986, 27: 642-647. 13. Bontkes HI, Veenendaal RA, Pefia AS, Goedhard JG, van Duijn W, Kuiper I, Meijer JL, Lamers CBHW: lgG subclass response to Helicobacterpylori in patients with chronic active gastritis and duodenal ulcer. Scandinavian Journal of Gastroenterology 1992, 27: 129133. 14. Talley N J, Neweli DG, Ormand JE, Carpenter HA, Wilson WR, Zinsmeister AR, Perez-Perez GI, Blaser MJ: Serodiagnosis of Helicobacter pylori: comparison of enzyme-linked immunosorbent assays. Journal of Clinical Microbiology 1991, 29: 1635-1639. 15. Morris A J, All MR, Nicholson GI, Perez-Perez GI, Blaser M J: Long term follow-up of voluntary ingestion of Helicobacter pylori. Annals of Internal Medicine 1991, 114: 662--663. 16. Kosunen TU, Seppiil~i 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. 17. Newell DG, Johnston B J, Ali MH, Reed Ph An enzyme-linked immunosorbent assay for the serodiagnosis of Campylobacter pylori-associated gastritis. Scandinavian Journal of Gastroenterology 1988, 23, Supplement 142: 53-57. 18. Faulde M, Schriider JP, Sobe D: Serodiagnosis of Helicobacter pylori infections by detection of immunoglobulin G antibodies using an immunoblot and enzyme immunoassay technique. European Journal of Clinical Microbiology & Infectious Diseases 1992, 11: 589-594. 19. Neweii DG: Human antibody responses to the surface protein antigens of Campylobacterpyloridis. Serodiagnosis and Immunotherapy in Infectious Disease 1987, 1: 209-217. 20. Rathbone BJ, Shires SE, Townsend C, Wyatt .11, Tompkins DS, Heatley RV, Losowsky MS: Serum Campylobacter specific IgG ELISA as a marker of colonisation in prevalence studies. In: Kaijser H, Falsen E (ed): Campylobacter IV. University of Goteborg, Goteborg, 1988, p. 182. 21. Dunn BE, Campbell GP, Perez-Perez GI, Blaser MJ: Purification and characterization of ureasc from Helicobacter pylori. Journal of Biological Chemistry 1990, 265: 9464-9469. 22. Gerstenecker R, Eschweiler B, Viigele H, Koch HK, Hellerich U, Kist M: Serodiagnosis of Helicobacter pylori infections with an enzyme immunoassay based on the chromatographically purified 120 kDa protein. European Journal of Clinical Microbiology & Infectious Diseases 1992, 11: 595--601.
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23. Apel I, Jacobs E, Kist M, Bredt W: Antibody response of patients against a 120 kDa surface protein of CampylobacterpyIori. Zentralblatt f~ir Bakteriologie, Mikrobiologie und Hygiene (A) 1988, 268: 271-276. 24. Evans DJ, Evans DG, Graham DY, Klein PD: A sensitive and specific serologic test for detection of Campylobacter pylori infection. Gastroenterology 1989, 96: 1004-1008. 25. Hirsehl AM, Pleschelle M, Hirschl MIt, Berger J, Stanek G, Roller ML: Comparison of different antigen preparations in an evaluation of the immune response to Campylobacterpylori. European Journal of Clinical Microbiology & Infectious Diseases 1988, 7: 570-575. 26. Goossens H, Glupczynski Y, Burelte A, van den Borre C, Bulzler JP: Evaluation of a commercially available second-generation immunoglobulin G enzyme immunoassay for detection of Helicobacter pylori infection. Journal of Clinical Microbiology 1992, 30: 176180. 27. Newell DG: Identification of the outer membrane proteins of Campylobacter pyloridis and antigenic cross-reactivity between C. pyloridis and C. jejuni. Journal of General Microbiology 1987, 133: 163-170. 28. Lee A, Logan SM, Trust T J: Demonstration of a flagellar antigen shared by a diverse group of spiral shaped bacteria that colonize intestinal mucus. Infection and Immunity 1987, 55: 828--831. 29. Andersen LP, Espersen F: Immunoglobulin G antibodies to Helicobacterpylori in patients with dyspeptic symptoms investigated by the western immunoblot technii:lue. Journal of Clinical Microbiology 1992, 30: 1743-1751. 30. Perez-Perez GI, Blaser MJ: Conservation and diversity of Campylobacter pyloridis major antigens. Infection and Immunity 1987, 55: 1256-1263. 31. wm Wulffen H: Low degree of relatedness between Campylobacter pyloridis and enteropathogenic Campytobacter species as revealed by DNA-DNR blot hybridization and immunoblot studies. FEMS Microbiology Letters 1987, 42: 129-133. 32. Newell DG, Slacey A: Antigens for the serodiagnosis of Campylobacter pylori infections. Gastroenterologie Clinique et Biotogique 1989, 13: 37B--41B. 33. wm Wulffen H: Methods of studying the immune response. In: Malfertheiner P, Ditschuneit H (ed): Helicobacter pylori, gastritis and peptic ulcel. Springer Verlag, Berlin Heidelberg, 1990, p. 137-140. 34. van den Oever HLA, Loffeld RJLF, Stobberingh EE: Usefulness of a new serological test (Bio-Rad) to diagnose Helicobacter pylori-associated gastritis. Journal of Clinical Microbiology 1991, 29: 283-286. 35. Milchell HM, Lee A, Berkowicz J, Borody I"." The use of serology to diagnose active Campylobacter pylori infection. Medical Journal of Australia 1988, 149: 604-609.
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36. Nonmra A, Stemmermann GN, Chyou PH, Kato I, Perez-Perez GI, Blaser M J: Heiicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii. New England Journal of Medicine 1991, 325: 1132-1136. 37. Parsonnet J, Friedmann GD, Vandersleen DP, Chang Y, Vogelman JH, Orentreich N, Sibley RK: Helicobacter pylori infection and the risk of gastric carcinoma. New England Journal of Medicine 1991, 325: 11271131. 38. Veenendaal RA, Pefia AS, Metier JL, Endtz HP, van der Est MMC, van Duijn W, Eulderink F, Kreuning J, Lamers CBH: Long term serological surveillance after treatment of Helicobacter pylori infection. Gut 1991, 32: 1291-1294. 39. Crablree JE, Mahony M J, Taylor JD, Healley RV, Littlewood JM, Tompkins DS: Immune responses to Helicobacter pylori in children with recurrent ab-. dominal pain. Journal of Clinical Pathology 1991, 44: 768-771. 40. Czinn SJ, Carr HS, Speck WT: Diagnosis of gastritis caused by Helicobacter pylori in children by means of an ELISA. Reviews of Infectious Diseases 1991, 13, Supplement 8: 700- 703. 41. Westblom TU, Madan E, Gudipati S, MidkilT BR, Czinn SJ: Diagnosis of Helicobacter pylori infection in adult and pediatric patients by using Pyloriset, a rapid latex agglutination test. Journal of Clinical Microbiology 1992, 30: 96-98. 42. Sobala GM, Crablree JE, Pentith JA, Rathbone BJ, Shallcross TM, Wyatt JI, Dixon MF, Heatley RV, Axon ATR: Screening dyspepsia by serology to Helicobacter pylori. Lancet 1991, 338: 94-96. 43. yon Wulffen H, Grote HJ, Kriimer-Hansen H: Serological screening for Campylobacter pylori in candidates for renal transplantation. Lancet 1987, i: 11401141. 44. Hirschl AM, Hirschl MH, Berger J, Rotter ML: Evaluation of a commercial latex teat for ~rological diagnosis of Helicobacter pylori infection in treated and untreated patients. European Journal of Clinical Microbiology & Infectious Diseases 1991, 10: 971-974. 45. Hoek FJ, Noach LA, Rauws EAJ, Tylgat GNJ: Evaluation of the performance of commercial test kits for detection of Helicobacter priori antibodies in serum. Journal of Clinical Microbiology 1992, 30: 15251528. 46. Przyklenk B, Bauernfeind A, Bornschein W, Gabor M: Evaluation of an IgG-ELISA-kit for diagnosing Heticobacter pylori associated gastroduodenal disease. Serodiagnosis and Immunotherapy 1990, 4: 263-269.
Vol. 11. No. 7
577
•Eur. J. Clin. Microbiol. Infect. Dis., July 1992, p. 577-582 0934-9723/92/07 0577-06 $ 3.00/0
An Assessment of Serological Tests for Detection of
Helicobacter pylori H. y o n Wulffen
Serological detection of Helicobacter pylori has been possible ever since the organism was first SUccessfully cultured by Marshall et al. in 1983 (1). Various methods have been employed including complement fixation (2, 3), agglutination (2), passive hemagglutination (4), indirect immunofluorescence (5), enzyme immunoassays (EIAs) (6-8) and immunoblot techniques (9, 10). All of these studies have shown that the vast majority of patients infected with Helicobacter pylori mount an easily detectable humoral immune response against this organism. Studies comparing the performance of older and more recent methods, for instance complement fixation compared with imrnunoblot (3), usually reveal superiority of the EIA and immunoblot methods over the other techniques. In the meantime a number of commercial tests, mostly based on the EIA technique, have appeared on the market. Two of these test kits have been evaluated in one of the three serological studies of Helicobacter pylori published in this issue of the journal (11). It can thus be expected that serological tests for Helicobacter Pylori will no longer be restricted to research purposes but will be included in the routine diagnostic work-up in patients with presumed gastrodUodenal pathology. For this reason it seems pertinent to reassess the current status of serological tests for Helicobacterpylori.
Immune Response Rathbone et al. (12) have shown that the immune response to Helicobacter pylori at the mucosal level is predominantly of the IgA type which is as could be expected in a chronic mucosal infection. Circulating antibodies against Helicobacter Pylori, however, are predominantly of the IgG class (12). IgG subclass analysis shows involvement of all four subclasses with a predominance Inslitut far Medizinisehe Mikrobiologie und Immunologie, Universitfitskrankenhaus Eppendorf, Martinistr, 52, 2000 Hamburg 20, Germany.
of the IgG1 and IgG2 subclasses (13). In this study of Bontkes et al. (13) patients with duodenal ulcers had a significantly higher IgG2 response than patients with chronic active gastritis without a duodenal ulcer. A systemic immune response of the IgA type usually seems to be less pronounced but if marked appears to indicate more severe inflammation (8, 14). For unknown reasons a small percentage of Helicobacter pylori-infected patients fail to mount a systemic IgG response and demonstrate IgA antibodies only. IgM antibodies are rarely found and seem to be of minor importance, however at present little information is available on acute Helicobacterpylori infection and subsequent seroconversion (15, 16).
Serological Techniques As mentioned above the full spectrum of serological techniques has been applied to Helicobacter pylori, the E I A and immunoblot methods having received the most attention. The immunoblot method has a very high sensitivity, one reason for this being the fact that any particular immunogenic protein concentrates in a very thin line on the nitrocellulose membrane. By using class-specific secondary antibodies the immune response can be differentiated with regard to the different immunoglobulin classes. As discussed below, it is frequently not so much the test method but the antigen preparation that determines the quality of a serological assay. The immunoblot technique is the best means for studying antigen preparations and their variations. For instance, comparative analysis of the protein profiles of different Helicobacter pylori isolates may help decide whether to use one or more strains as antigen. Immunoblot analysis may also reveal the presence or absence of crossreacting proteins in different preparations of the antigen. Thus, no matter what method is used for serological tests, the immunoblot is an excellent technique for quality control of an antigen
578
preparation. However, problems may also arise when using the immunoblot method as reference for other techniques. In complex mixtures such as a whole cell lysate or a sonicate where a very dense set of reactive bands results, it may be difficult to identify a particular protein band. Furthermore, depending on the quality and proportion of polyacrylamide and the method of transfer used, not all proteins of interest may be transferred to the nitrocellulose during the blotting procedure. Since the antigen samples are usually boiled in sodium dodecyl sulfate (SDS) and other denaturing agents, some of the immunodominant epitopes may no longer be recognized. Apart from that, the immunoblot technique is quite labor-intensive and thus not suitable for testing large numbers of sera. Moreover, it only allows semiquantitative interpretation of results. The EIA, on the other hand, is easy to perform and therefore well suited for screening purposes. It possesses comparable sensitivity, may also be used for detection of class-specific antibodies, and - a major advantage - results may be expressed quantitatively It is not necessary to use end-point titrations for this purpose. Results may be expressed as EIA units (U/ml) based on serial dilutions of an internal standard serum (8). Alternatively, the concentration of anti-Helicobacter pylori antibodies can be measured by using a standard curve for human immunoglobulin (17), the results being expressed as the amount in micrograms of bound immunoglobulin per millilitre. An interesting variation of the EIA is the transferable solid phase EIA technique (TSP-EIA) or pin-plate technique employed by Faulde et al. (18) which is described in this issue of the journal. It allows synchronisation of processing during all incubation steps thereby eliminating intra-assay variations caused by time delays in processing between the first and last wells of a plate.
Choice of Antigen Early studies employed crude antigens such as whole cells or whole cell sonicates with acceptable results (2, 6, 12). Improved performance values were reported using partially purified antigens such as acid glycine extracts (7, 8, 19) or an ultracentrifuged supernatant from a whole cell sonicate (20). More recently, attempts have been made to use purified antigens to further reduce cross-reactivity and improve specificity. When
Eur. J. Clin. Microbiol. Infect. Dis.
highly purified antigens such as the Helicobacter pylori urease (21) or the 120 kDa protein, as described by Gerstenecker et al. (22) in their paper in this issue of the journal, have been employed, specificity was indeed increased. However, not all Helicobacterpylori-infected patients demonstrated antibodies against these antigens, and thus the sensitivity was lower. For the 120 kDa protein this can be partly explained by the fact that not all infecting Helicobacter pylori strains exhibit this particular protein (23). It has therefore been suggested that two or more purified antigens be combined in one EIA. Evans et al. (24) describe an assay which only employs high molecular weight cell-associated proteins (HM-CAP) and which apparently has both high sensitivity and specificity. Hirschl et al. (25) reported similar results using high molecular weight proteins between 80 and 130 kDa. One of the commercially available test kits (Cobas Core Anti-Helicobacter pylori EIA, Hoffmann LaRoche, Switzerland) combines a number of fast protein liquid chromatography (FPLC) fractions including the native urease enzyme and performs favorably according to Goossens et al. (26). Other manufacturers give very little information on their antigen preparations, but certainly some of them use partially purified antigens such as acidglycine extracts. The importance of using highly purified antigens is still a matter of controversy. The main crossreactivity reported is between the flagellar proteins of Helicobacter pylori and Campylobacterjejuni in the 54-59 kDa region (27, 28). However, in a recent immunoblot study conducted by Andersen et al. (29), serological cross-reactions with other bacteria were very limited. In particular, in absorption studies only one minor reactive band of approximately 100 kDa was absorbed from a Helicobacterpylori-positive serum pool by Campylobacterjejuni and Campylobacter coli strains. Talley et al. (14) compared four antigen preparations, namely a sonicate, an acid-extractable surface antigen, an acid-extractable antigen from an aflagellate variant (to reduce cross-reactivity with Campylobacterjejuni), and a FPLC-fractionated urease-containing antigen. They found that the diagnostic value of the different EIAs was highly comparable, and that the crude antigens performed as well as the more purified antigens, the FPLC-purified antigen displaying the lowest sensitivity. However, crude antigens usually give higher backgrounds which may make it difficult to establish optimal cut-off values to discriminate between low positive and
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true negative results. Therefore some sort of purified multi-component antigen is certainly desirable to achieve both high sensitivity and specificity. The question arises whether the antigen preparation should be derived from one or more strains of Helicobacter pylori. Several studies (27, 29-3I) have shown that there are only minor differences in antigenic composition between different isolates of Helicobacter pylori. Nevertheless, it has also been reported that sera from some Helicobacter pylori-infected patients can have a negative reaction with antigen from one strain but a Positive reaction with antigen from other strains (32).
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under study as impressively demonstrated in the recent study of Goossens et al. (26). These authors evaluated the Cobas Core AntiHelicobacter pylori E I A (Hoffmann-LaRoche, Switzerland) in 855 Belgian and 257 Mediterranean patients in whom upper gastrointestinal endoscopy was clinically indicated. They obtained a very good negative predictive value of 96 % in the Belgian population where there was an age-dependent prevalence of Helicobacter pylori infection ranging from 34 to 53 %. In the Mediterranean group, on the other hand, where there was a high prevalence of Helicobacterpylori infection of 87 %, they achieved a rather poor negative predictive value of 64 %. Specificity of the assay also dropped from 95 % in the Belgian group to a modest 70 % in the Mediterranean group.
Serological assays are usually evaluated on the basis of performance criteria such as sensitivity, Specificity, and positive and negative predictive Values. To obtain these data, a set of clearly defined positive and negative reference sera is needed. This raises the issue of the reference method. For the purist among the microbiologists culture of the organism should serve as reference. However, the distribution of Helicobacter pylori and the related type B gastritis in the stomach rnucosa is irregular so that the question arises as to how many biopsies and cultures are needed to obtain 100 % for the proposed reference method. Depending on the quality of the reference method, a serological technique may appear to perform inferiorly even if in fact it is more acCUrate. Whether a biopsy urease test, urea breath tests, microscopic detection of the typical organism or even the histopathologist's diagnosis of type B gastritis come into question as reference method for serological tests probably depends on the quality of the reference methods available in a given situation. Using the same acid glycine extract as antigen for an IgG EIA, our team arrived at quite different performance values depending on the reference method employed. Overall, we obtained the best values using a 14C-urea breath test as reference (33).
As the performance values relate to the chosen cut-off value and the prevalence of the infection in the patient population, it may be advisable to re-evaluate a test before use if it has originally been tested in a different patient population. This also holds true for commercial test kits, as has been clearly demonstrated by van den Oever et al. (34). When these authors evaluated the commercial GAP test (Bio-Rad Laboratories, USA) following the manufacturer's instructions, they achieved values for sensitivity, specificity, the positive predictive value and negative predictive value of 32.7 %, 100 %, 100 % and 58.9 % respectively. They then established a receiver operating characteristic (ROC) curve which involves plotting true-positive rates against false-positive rates for various possible cut-off values. The cut-off value whose R O C curve encompasses the largest area is considered to be the most accurate. By establishing the optimal cut-off value of the G.A.R test for their population in this manner, they arrived at values for sensitivity, specificity, the positive predictive value and negative predictive value of 87.8%, 91.3 %, 95.6 % and 77.8% respectively, which seems more feasible. Another example of use of such R O C curves for determination of the optimal cut-off value and for comparing the quality of different assays is given in the study of Aguirre et al. (11) presented in this issue of the journal.
Care is necessary in evaluating different EIAs solely on the basis of performance values when these values have been derived from different Populations. The negative predictive value, in particular, is highly dependent on the actual prevalence of the infection in the population
While possible cross-reactions may not be relevant in one population, they may very well be of importance in a different population. This has been shown by Mitchell et al. (35) who employed the same sonicated antigen in an EIA for screening both a Sydney population and sera from a
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Papua New Guinean population. While absorption of sera with Campylobacter jejuni had little effect on the results in the Sydney patients, this proved to be a crucial step in the Papua New Guinean population. Without the absorption step the Helicobacter pylori infection rate in Papua New Guineans appeared to be 56 %, whereas use of the absorption step reduced this rate to 25 %. Thus, in populations with a high prevalence of anti-Campylobacter jejuni antibodies these findrags should be taken into account.
Applications of Serological Tests Serological tests provide a practical and non-invasive means of assessing Helicobacter pylori infection in an individual patient or in a large population. The only non-invasive alternative is urea breath tests, however these require special equipment, time for collection of breath samples, and use of either radioactive material or tracers that can only be measured with mass spectrometers. • One of the main fields of application of serological tests to detect Helicobacterpylori is epidemiology. In gastric cancer epidemiology such tests have become an indispensable tool, the best evidence we have today of an association between chronic Heticobacter pylori infection and gastric cancer being obtained in serologic screening of high-risk populations (36, 37). Another important indication for such serological investigations is in the long-term follow-up of Helicobacter pylori eradication therapy. Several authors could show that significant falls in antibody titers usually occur within about six months after successful eradication therapy (8, 16, 38). Again, IgG titers give the best results. Peptic ulcer disease is not a rare finding in childhood, and abdominal pain even less so. Use of upper gastrointestinal endoscopy, however, is usually avoided or delayed in these patients as, among other things, it can involve general anaesthesia. Serological tests for Helicobacter pylori might be very helpful in this patient population for screening. Again, this is a population where the mode of evaluation may have to be changed. Thus it has been suggested that cut-off values may have to be adjusted in pediatric patients (39-41). Suggestions have also been made to employ serological tests for Helicobacter pylori in adult patients to lower the endoscopy load and thereby
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costs. By using a screening strategy based on age, serological tests for Helicobacter pylori and documentation of use of non-steroidal anti-inflammatory drugs (NSAIDs), Sobala et al. (42) demonstrated that they might have reduced the endoscopy workload by 23.3 % and would have had a sensitivity in the detection of peptic ulcers of 97.4 %. No case of malignant disease would have been overlooked although 6 of 192 cases of peptic ulcer in a group of 842 patients would not have been detected. A policy of symptom-based screening in dyspeptic patients under the age of 45 would in fact have been less sensitive than the proposed screening based on serological tests for Helicobacter pylori whereby only those patients underwent endoscopy who were either sero-positive for Helicobacter pylori, taking NSAIDs or aged 45 years or more. The age limit of 45 was chosen to minimise the risk of overlooking cases of upper gastrointestinal malignancy. Serological tests for Heticobacter pylori might also be effectively employed for screening certain high-risk patients as we demonstrated in a retrospective study of patients before and after renal transplantation (43). Such patients may have been infected with Helicobacter pylori for many years without overt disease until an event such as a transplantation compromises their immune system, including the mucosal defense mechanisms of the stomach and duodenum. Early serodiagnosis and appropriate therapy aimed at eradication of Helicobacter pylori might possibly prevent these patients from developing peptic ulcers and gastrointestinal hemorrhage.
Conclusions Serological tests provide an easy and non-invasive means of detecting Helicobacter pylori infection. Testing for IgG antibody usually suffices; testing for lgA may be complementary in some instances. A number of commercial assays are now available on the market, and such tests will undoubtedly become part of the routine diagnostic work-up. However, the performance values (sensitivity, specificity, predictive values) reported for these tests have rarely been above 90 % which is not very satisfactory (11, 26, 34, 4446). Furthermore, the optimal antigen composition has not yet been determined. Cut-off values proposed by the manufacturers are often not satisfactory and should be re-evaluated for a particular population. It must also be taken into ac-
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count that performance values are closely interrelated to the prevalence of the infection in the population under study. Serological tests for Helicobacter pylori are particularly useful for monitoring the results of eradication therapy. They may also be useful for screening certain patient populations to help decide whether to perform endoscopy provided certain rules are strictly adhered to. It is not advisable, however, to base the diagnosis and treatment of patients solely on positive serological tests for Helicobacter pylori, a practice which is unfortunately already gaining ground.
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