1556-6811/08/$08.00⫹0 doi:10.1128/CVI.00165-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Evaluation of a Commercial Immunoblot, Helicoblot 2.1, for Diagnosis
of
Helicobacter pylori
Infection
䌤
Lea Veijola,
1,2* Aino Oksanen,
1Pentti Sipponen,
3and Hilpi Rautelin
2,4Herttoniemi Hospital, City of Helsinki, Helsinki, Finland1; Department of Bacteriology and Immunology, Haartman Institute,
University of Helsinki, Helsinki, Finland2; Biohit PLC Diagnostics, Helsinki, Finland3; and HUSLAB,
Helsinki University Central Hospital Laboratory, Helsinki, Finland4
Received 11 May 2008/Returned for modification 28 July 2008/Accepted 21 September 2008
The best method to diagnoseHelicobacter pyloriinfection in different clinical situations is controversial. The aim of the study was to assess the performance of a commercial immunoblot, Helicoblot 2.1. The study comprised 215 patients, who were grouped according to the presence ofH. pyloriinfection (assessed by two gastroscopies including histology with a median interval of 7.1 years, enzyme immunoassay [EIA]-based serology, and history of previousH. pylori infections and eradication therapies) into four categories: noH. pylori infection ever, previous infection, ongoing infection, and EIA seropositivity as the only marker of a possible previous infection. The sensitivity of Helicoblot 2.1 to show an ongoing or previousH. pyloriinfection was 100% and 92%, respectively. Helicoblot 2.1 was negative in only 80% of individuals with no evidence of present or previous infection but in 96% of patients 50 years of age or younger. The current infection marker of the immunoblot was positive in 49% of patients with successfulH. pylorieradication therapy. After successful eradication therapy, Helicoblot 2.1 sustained positive results in 87% of patients, and CagA positivity was detected in 87% of patients with follow-up samples for more than 10 years after therapy. Helicoblot 2.1 is a sensitive and, among patients of ages 50 years or younger, a specific test in the primary diagnosis ofH. pylori
infection. However, it does not discriminate between past and current infections. It can be used in epidemio-logical studies assessing the role ofH. pyloriin different late sequelae.
Helicobacter pyloriinfection is clearly associated with peptic
ulcer disease, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma (17). None of the available diag-nostic methods forH. pyloriinfection are ideal (23). The inva-sive methods, such as rapid urease test, histology, and culture, have high specificity but low sensitivity in the presence of atrophic gastritis and during proton pump inhibitor (PPI) ther-apy (22). The same is true for the noninvasive urea breath and stool antigen tests. Serology is recommended to be used in atrophic gastritis and during PPI therapy, but a single serum sample does not usually differentiate the past and ongoing infections. Even 30% of patients had elevated antibodies of the immunoglobulin G (IgG) class still after 5 years of successful eradication therapy (43).
The true impact ofH. pylorion gastric cancer incidence is not really known (21, 31). Patients with advanced atrophic gastritis have the highest cancer risk (39), but the best method for diagnosing a pastH. pylori infection in gastric atrophy is debatable. CagA antibodies are thought to best reflect the past infections and were shown to sustain for longer than otherH.
pyloriantibodies in a follow-up of 32 months (40). However,
the specificity of CagA antibodies has been challenged (38). Furthermore, the role ofH. pyloriin a severe autoimmune type of gastric atrophy also remains unresolved (1).
Several immunoblotting methods for the diagnosis of H.
pyloriinfection are available, but the Helicoblot test is probably
the most studied (11). The previous studies of the performance
of the Helicoblot test in the diagnosis of an ongoingH. pylori
infection have shown variable results. In children, the sensitiv-ities of Helicoblot 2.0 (32, 34) and Helicoblot 2.1 (26, 28) varied between 95.5 and 100% and 80 and 98.6%, respectively, and the specificities between 85.7 and 88% and 87.1 and 100%, respectively. In adults, Helicoblot 2.1 showed sensitivities of 93.4 to 99% and specificities of 88 to 98% (12, 18, 25, 30, 44). After eradication therapy, the seroreversion rates have been low, less than 10% (14), and the current infection marker (CIM) has been shown to be unreliable (18). The accuracy of Helicoblot 2.1 in determining the CagA or VacA status com-pared to that of the genotyping has been variable, and in many studies, the use of the test for this purpose has not been recommended (8, 15, 30).
The aim of this study was to evaluate the performance of the commercial immunoblot Helicoblot 2.1 in a series of patients, both in the primary diagnosis ofH. pyloriinfection and with follow-up data available for about 7 years after eradication therapy. We also evaluated the role of CagA antibodies in the diagnosis of a pastH. pyloriinfection.
Helicoblot 2.1 was a sensitive and specific test in the primary diagnosis ofH. pyloriinfection, especially in younger patients. It had a low discrimination value in the assessment of the success ofH. pylorieradication therapy. BecauseH. pylori an-tibodies detected by Helicoblot 2.1, especially those to CagA, remained for years after successful eradication therapy, immu-noblotting seemed to be the most sensitive method to detect a
pastH. pyloriinfection.
MATERIALS AND METHODS
Patients.During the 2-year period from 1 January 2004 to 31 December 2005, a total of 345 consecutive patients with at least one previous upper
gastrointes-* Corresponding author. Mailing address: Herttoniemi Hospital, Kettutie 8, 00800 Helsinki, Finland. Phone: 358-505564367. Fax: 3589-19126382. E-mail: lea.veijola@helsinki.fi.
䌤Published ahead of print on 30 September 2008.
1705
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tinal endoscopy a median of 7.1 years earlier underwent gastroscopy, and 235 of the patients also gave serum samples. Of the 235 patients, 94 had no signs of a
previousH. pyloriinfection either in history (no known eradication therapy ever),
in a previous gastroscopy including histology (a median of 7.4 years earlier; report available for 78 patients), or in laboratory tests (results of previous diagnostic tests when available or the tests performed in the present study). An
additional 84H. pylori-negative patients had been successfully treated forH.
pyloria median of 6.6 years earlier (the exact eradication date was unavailable for
five patients). An ongoingH. pyloriinfection was verified by histology in 26
patients, and one additional patient with noH. pyloridetected by histology had
active gastritis and positiveH. pyloriculture and serology. Seven of these 27
patients had had unsuccessful eradication therapies earlier. In an additional 20
patients, a formerH. pyloriinfection could not be excluded; 14 patients had
severe corpus atrophy (grade 2 to 3 according to the updated Sydney classifica-tion), and 6 other patients had a history of duodenal ulcer (two had had gastric
resection, and three had still-elevatedH. pyloriantibody levels). Although they
had neither knownH. pylorieradication therapy nor signs of a currentH. pylori
infection in histology, these particular 20 patients were excluded from the study.
Ten more patients had elevatedH. pyloriantibodies (six in IgG class, and six in
IgA class). They had neither known eradication therapy in their history nor signs ofH. pyloriinfection in gastroscopies performed a median of 6.1 years earlier, but
previousH. pyloriinfection and unintentional eradication of the infection could
not be excluded. They were included in the study but assessed separately. Thus, the final study population consisted of 215 patients (158 females and 57 males; median age, 64 years). The patient characteristics in the different groups are presented in Table 1.
All patients gave their written informed consent, and the study was approved by the local ethics committee.
Histology. During the gastroscopy of the present study, two biopsies were taken from both antrum and corpus for histology. Biopsies were stained with hematoxylin and eosin, Alcian blue (pH 2.5)-periodic acid-Schiff, and modified Giemsa stains and assessed according to the updated Sydney System by one experienced pathologist (P.S.) unaware of the identity of the samples (3).
Serum tests.The serum specimens of the 215 patients were stored at⫺20°C
until examined. Both IgG and IgA antibodies toH. pyloriwere determined by an
in-house enzyme immunoassay (EIA) method as described earlier (27). The sensitivities and specificities compared to the histology results have been 99% and 93% for the IgG antibodies and 64% and 98% for IgA antibodies, respec-tively (27).
The immunoblot test was performed, and the results were assessed by one laboratory assistant according to the manufacturer’s instructions. The
interpre-tation criteria for anH. pyloriseropositive sample were as follows: (i) fulfilling
the criteria for CagA positivity (namely, the presence of a 116-kDa CagA band in combination with CIM, with the 30-kDa UreA band and the 19.5-kDa band, or with the 89-kDa VacA band, the 37-kDa band, or the 35-kDa band); (ii) the presence of any of the bands at 89 kDa, 37 kDa, or 35 kDa; and (iii) the presence of both the bands at 30 kDa and 19.5 kDa. The intensity of the bands was graded with a naked eye from 1 to 3, grade 1 being defined as a very faint barely visible band.
RESULTS
The overall performance of Helicoblot 2.1 in patients with no knownH. pyloriinfection, those with an ongoingH. pylori
infection, those with a pastH. pyloriinfection, and those with
TABLE 1. Characteristics of the patients grouped according toH. pyloristatus
Characteristica
Value for patients with:
No known infection
(n⫽94)
Previous infection
(n⫽84)
Ongoing infection
(n⫽27)
ElevatedH. pylori
antibodies only
(n⫽10)
Median age (yr) 61.3 65.6 64.9 69.3
Age range (yr) 24–85 28–87 42–82 26–79
Median time between gastroscopies (yr) (no. of patients with data available)
7.4 (78) 6.7 (74) 9.9 (18) 6.1 (9)
Female/male (%) 68 (72)/26 (28) 66 (79)/18 (21) 19 (70)/8 (30) 5 (50)/5 (50)
No. of patients with following main endoscopy indications (%):
Abdominal pain 31 (33) 37 (44) 10 (37) 4 (40)
Reflux 28 (30) 17 (20) 4 (15) 4 (40)
Dyspepsia 11 (12) 8 (10) 6 (22) 0
Anemia or diarrhea 7 (7) 2 (2) 1 (4) 1 (10)
No. of patients with following macroscopic findings (%):
Erosive esophagitis/Barrett 10 (11)/0 12 (14)/1 (1) 3 (11)/0 1 (10)/0
Erosive gastritis/ulcer 8 (9)/1 (1) 9 (11)/1 (1) 2 (7)/3 (11) 2 (20)/0
Duodenal erosion 2 (2) 1 (1) 0 1 (10)
No. of patients with following histologic findings (%):
Chronic gastritis 9 (10) 29 (35) 27 (100) 1 (10)
Atrophic corpus gastritis 1 (1) 3 (4) 5 (19) 0
No. of patients with PPI use on daily basis (%) 17 (18) 13 (15) 1 (4) 0
No. of patients with no PPI use (%) 4 (4) 5 (6) 2 (7) 0
No. of patients with NSAID use (%):
Less than once a mo 15 (16) 11 (13) 4 (15) 1 (10)
More than once a wk 17 (18) 15 (18) 3 (11) 1 (10)
a
NSAID, nonsteroidal anti-inflammatory drug.
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elevated H. pylori EIA-based antibody levels as the only marker of a possible previous infection is presented in Table 2. Helicoblot 2.1 showed a high sensitivity, 100%, in patients with a current infection and a moderate sensitivity, 92%, for the detection of a past infection. If the presence of the 116-kDa (CagA) band alone had been regarded as a sign of seroposi-tivity (against the criteria of the manufacturer), the sensiseroposi-tivity of the test for detecting a past infection would have been 89%. The specificity of the test was somewhat disappointing, as 20% of the patients with no knownH. pyloriinfection gave false-positive reactions. However, when this particular patient group was further subdivided according to age (Table 3), the Heli-coblot test results showed good specificity (96%) in the younger age group (ⱕ50 years); the only false-positive patient also hadH. pyloriantibodies of the IgG class near the upper limit (titer, 600) of the cutoff value (titer, 700). Many of the patients older than 50 years of age had false-positive immuno-blot results (18 out of 71 [25%]). On the contrary, the three patients with elevatedH. pyloriantibodies in EIA results but negative results by Helicoblot 2.1 were 26, 45, and 56 years of age.
The persistence of antibodies to differentH. pyloriantigens in Helicoblot 2.1 after successful eradication therapy is pre-sented in Table 4. In 22 of the 53 patients (42%) for whom more than 5 years have passed since successful eradication, the CIM was still positive. The CagA antibodies sustained positive results in many patients even⬎10 years after eradication. The exclusion of the faint reactions to CagA antigen decreased the sensitivity of the immunoblot by 7 to 20% at all time intervals, and the proportion of the weak reactions did not increase with the time elapsed from the eradication date. Reactions to the VacA and UreA antigens decreased faster after eradication than those to the CagA antigen.
DISCUSSION
Helicoblot 2.1 showed high sensitivity in detectingH. py-lori antibodies in all but one presently H. pylori-infected subjects and very high specificity (96%) in young patients (ⱕ50 years old). Thus, Helicoblot 2.1 could be used as a confirmatory test in controversial clinical situations. How-ever, antibodies to the CIM band poorly differentiated the patients with past and ongoing infections, as half of the patients with successful eradication therapy showed a posi-tive CIM band. The antibodies detected by the immunoblot persisted, in most cases, for years even after successful erad-ication therapy, which enables the detection of past infec-tion in epidemiological studies.
The well-characterized patients with follow-up data avail-able for years with no knownH. pylori infection detected in either previous or present gastroscopy or laboratory tests and no known eradication therapy seemed to show a high preva-lence of previous infections, according to the immunoblot anal-ysis results. However, this was the case only in the older pa-tients. Helicoblot 2.1 was actually very specific (96%) in the group of patients 50 years of age or younger. The prevalence of
H. pyloriinfection is rapidly declining in Finland (33) and in
other developed countries in young-age cohorts. As the posi-tive predicposi-tive value of the test is strongly dependent on the prevalence of the disease, the proportion of false-positive test results relative to the true-positive results may be a problem with many of the noninvasiveH. pyloritests (42). Helicoblot 2.1 could be a confirmatory test for the discrepant results in the younger age group.
The interpretation of the false-positive Helicoblot 2.1 results in the age group older than 50 years is difficult. It is tempting to speculate that the false-positive test results could actually be because of remnants after a spontaneous disappearance of the infection. Another possibility is that, for some reason, Heli-coblot 2.1 has low specificity in older age groups, an unresolved problem also contemplated previously (9). Studies comparing the accuracy of the Helicoblot test with that of PCR methods in determining the CagA status have given discrepant results (15, 25, 30, 47), and the reliability of the immunoblot test to verify the CagA status has actually been challenged (37). In a study assessing the gastric histology, most patients with
CagA-TABLE 2. Numbers of patients, grouped according toH. pylori
status, with positive bands in Helicoblot 2.1 resultsa
Positive reactions
No. of patients (%) with:
No known infection
(n⫽94)
Previous infection
(n⫽84)
Ongoing infection
(n⫽27)
Only elevated
H. pylori
antibodies
(n⫽10)
H. pyloripositive 19 (20) 77 (92) 27 (100) 7 (70)
H. pyloriand CIM positive
5 (5.3) 41 (49) 22 (81) 5 (50)
CagA 116-kDa band positive
24 (26) 75 (89) 26 (96) 8 (80)
With faint bands excluded
19 (20) 62 (74) 25 (93) 6 (60)
CagA positivityb 16 (17) 73 (87) 26 (96) 7 (70)
VacA 89-kDa band positive
14 (15) 62 (74) 26 (96) 7 (70)
UreA 30-kDa band positive
15 (16) 60 (71) 27 (100) 4 (40)
UreB 61-kDa band positive
91 (97) 84 (100) 27 (100) 10 (100)
HSP 58-kDa band positive
90 (96) 84 (100) 27 (100) 10 (100)
aThe intensity of the band was assessed by naked eye as faint if the band was
barely visible.
bCagA positivity was determined according to the manufacturer’s criteria
(also, the faint bands are included).
TABLE 3. Numbers of patients with no knownH. pyloriinfection showing positive Helicoblot 2.1 results and
grouped according to age
Immunoblot test result
No. of patients (%) who are:
ⱕ50 yr old
(n⫽23) ⬎
50 yr old
(n⫽71)
H. pyloripositive 1 (4.3) 18 (25)
H. pyloriand CIM positive 1 (4.3) 4 (5.6)
CagA 116-kDa band positive
3 (13) 21 (30)
With faint bands excluded
2 (8.7) 17 (24)
CagA positivitya 1 (4.3) 15 (21)
VacA 89-kDa band positive 1 (4.3) 13 (18) UreA 30-kDa band positive 2 (8.7) 13 (18)
aCagA positivity was determined according to the manufacturer’s criteria
(also, the faint bands are included).
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positive serum samples were suggested to have real previous infections (49). In our study of 215 patients (excluding the patients with severe corpus atrophy and duodenal ulcer his-tory, because of their ambiguousH. pyloristatus), 104 patients had no knownH. pyloriinfection or eradication therapy earlier and noH. pyloriin histology in the present study. Ten of these 104 patients had positiveH. pyloriEIA serology (IgG and/or IgA antibodies elevated), but 23 (22%) were CagA positive according to the Helicoblot 2.1 criteria (the CagA band was positive in 32 more patients [31%]). These results are in accordance with studies suggesting a much higher sponta-neous disappearance rate of theH. pyloriinfection (19) than has previously been anticipated to happen (4% in 21 years) (16). If these particular patients are at an increased risk for
H. pyloriinfection, associated long-term sequelae remains to
be studied.
In our study, CagA antibodies indeed sustained for longer after successful eradication than did antibodies to other anti-gens, in accordance with some previous studies (18, 40). The prevalence of CagA positivity, however, slightly declined with time, as the presence of antibodies to the other antigens, needed to fulfill the manufacturer’s criteria for CagA positiv-ity, declined more rapidly. Antibodies solely to the 116-kDa CagA antigen (not fulfilling the criteria for CagA seropositivity according to the manufacturer) were detected in 11 (5%) pa-tients of the whole 215-patient study population, and 2 of them were known to have been previously infected withH. pylori. In studies assessing the impact ofH. pylori infection on gastric cancer risk (6, 5, 10, 29, 35, 41, 48, 50) or on the development of atrophic gastritis (1, 4, 7, 24, 36), the different definitions of the criteria for CagA positivity as a sign of a previous contact
withH. pylorimay give different risk estimations. In our study,
among patients for whom it had been more than 5 years since successful H. pylori eradication, the positive EIA serology found only 18 (34%) of the 53 patients with verified pastH.
pyloriinfection, but the immunoblot detected 45 (85%)
sub-jects, if CagA positivity according to the manufacturer’s criteria were used, and even more, 47 (89%) subjects, if antibodies solely to CagA antigen had been considered; the specificities were 83% and 74%, respectively. This high sen-sitivity is in accordance with a previous study (30), which, however, showed very low specificity when the interpreta-tion criteria were changed. In our study, Helicoblot 2.1 had good sensitivity compared to that of EIA serology in detect-ing a past infection also when the manufacturer’s criteria were followed.
It has been suggested that faint positive reactions to CagA
antigen could be false positive (38). However, in our study, the exclusion of patients with antibodies giving only faint reactions to CagA did not significantly increase the overall performance of the test. The specificity of the test only increased from 74% to 80% in patients with no knownH. pylori infection, but in patients with previousH. pyloriinfection, the exclusion of faint bands dropped the sensitivity from 89% to 74%. The unreli-ability of the CIM band for differentiating ongoing and pastH.
pyloriinfections was in accordance with an earlier study
show-ing positive CIM in 80% of patients 3 years after successful eradication therapy (18). Furthermore, in our study, this could be confirmed in patients with substantially longer follow-up periods.
The heat shock protein (HSP) 58-kDa band (13, 46) has been associated with different gastroduodenal diseases, in par-ticular, gastric atrophy (2, 20). It has also been associated with intensity of chronic inflammation in the antrum (45). In our study, antibodies to the 58-kDa antigen were present in 96% of the patients with no known previousH. pyloriinfection.
In conclusion, Helicoblot 2.1 seems to be a sensitive and specific test in the primary diagnosis of H. pylori infection, especially in younger patients. It has, however, a low discrim-ination value in the assessment of the success of H. pylori
eradication therapy. BecauseH. pyloriantibodies detected by Helicoblot 2.1, especially those to CagA, remain for years after successful eradication therapy, immunoblotting seems to be the most sensitive method to detect a pastH. pyloriinfection. Future studies are needed to clarify the impact of Helicoblot 2.1 as a screening method to define the importance of a pastH.
pyloriinfection on the increased risk of gastric cancer or
atro-phic gastritis.
ACKNOWLEDGMENTS
Our deepest appreciation goes to the late Pirjo Kosonen for her skillful assistance.
The study was partly supported by a grant from City of Helsinki to L.V. L.V., A.O., and H.R. have no conflicts of interest. P.S. is a scientific advisor and a shareholder of Biohit Plc, a Finnish com-pany that produces and markets laboratory pipettes and laboratory tests.
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