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Discrepancy between Helicobacter pylori stool antigen assay and urea breath test in the detection of Helicobacter pylori infection
ALIMENTARYTRACT
DIGEST LIVER OIS 2000;32:285-90
Discrepancy between Helicobacter pyiori stool antigen assay and urea breath test in the detection of Helicobacter pylori infection G. L. P. S. C. A. A.
Masoero Lombard0 Della Monica’ Vicari Crocilk2 Duglio2 Pera
Background. The reference diagnostic methods available for detection of Helicobacter pylori infection are either invasive [histology] or expensive and highly sophisticated [Urea Breath Test]. A new enzyme immunoassay, which can be easily performed in any laboratory has been developed to detect Helicobacter pylori in stool specimens [HpSA - Meridian Diagnostics, Cincinnati, USA]. Aim of the study was to compare HpSA to Urea Breath Test. Patients and methods. A total of 125 patients (52 never treated for Helicobacter pylori infection and 73 after Helicobacter pylori eradication therapy] referring to our Department, underwent both tests within two weeks. Results. Contrasting results between the two tests were found in 30% of cases: in 19% of the untreated patients and in 37% of the treated patients [p
Digest
Liver
Key words: test
Dis 2000;32:295-90 coccoid
forms
of
Helicobacter py/wi;
Helicobacter py/ori
stool
assay;
urea
breath
The best technique for the diagnosis of Helicobacter pylori (H. pylori) infection is yet to be identified, and a universally accepted gold standard is still lacking. Diagnostic methods may be invasive (endoscopic) or non-invasive (non-endoscopic). Among the former, histological examination of multiple biopsy specimens is accepted as reference method, although it includes disadvantages related to all biopsy methods, due to sampling errors and observers’ variations. Among the non-invasive methods, the 13C -Urea
Discrepancy
between HpSl and UBT
Breath test (UBT) is considered the most reliable in assessing H. pylori infection both before and after eradication ‘; when carried out at least four weeks after the end of treatment, it would appear to be as accurate as biopsy-based tests, perhaps even more * 3. Serological tests vary greatly in accuracy and appear to be less reliable than UBT, especially in the post treatment assessment 4. However, since the UBT-technique requires highly sophisticated and expensive equipment along with specialised staff - not universally available - the demand for a simpler, equally reliable and cost-effective test for H. pylori detection is persistently high. Recently, a new enzyme immunoassay test 5 has been developed to detect H. pylori in stool specimens, namely Meridian Diagnostics, HpSA, Cincinnati, USA; this test has the advantage that it is easy to perform in any laboratory thanks to the possibility of the simple visual or spectrophotometric interpretation. Aim of our study was to compare HpSA to 13C-UBT in a group of patients referring to our GI unit. UBT was adopted as gold standard.
Patients and methods Patients A total of 125 consecutive patients, either dyspeptic or with a previous history of peptic disease (68 females and 57 males; mean age 52 years, range 15-79) referring to our GI Unit for H. pylori assessment by means of UBT, were included in the study. Of these, 52 subjects had never been submitted to any H. pylori eradication therapy; in 73 cases, the indication for UBT was to evaluate success of treatment. The time elapsing between the end of treatment and the test itself ranged from 2 to 212 months. Exclusion criteria were: use of antisecretory drugs, bismuth-based compounds or antibiotics within 2 months prior to the two tests. Fresh stool specimens were collected within 1 week of UBT and immediately stored at -20°C until the running of the assay. Methods Urea breath test The test was performed according to the European protocol 6. Fasting subjects were given a standard meal (200 ml of full cream milk or orange juice) to delay gastric emptying, followed by a solution of 100 mg of urea isotopically labelled with 13C. Duplicate breath samples were collected in basal conditions and 30 minutes after ingestion of 13C-Urea. The 13C enrichment of samples was determined by isotope ratio mass spectrometry (ABCA- Europa Scientific, Crewe, UK). A test was considered positive when the difference between the ‘“C-enrichment at 30 286
minutes over the baseline (DOB-30) was >4 per mil. In our laboratory, where UBT has been routinely performed for 3 years, the sensitivity (SS) and specificity (SP) of the test, evaluated using histology as gold standard, are 92% and 97%, respectively. H. pylori stool assay Faecal H. pylori detection was performed by means of a commercially available kit (Premier Platinum HpSA - Meridian Diagnostics, Cincinnati, USA). The test uses polyclonal anti-H. pyZori antibodies adsorbed to microwells. Diluted stool samples and a peroxidase conjugated polyclonal antibody are added to the wells and incubated for 1 h at room temperature. Substrate is added and incubated for 10 minutes. Colour develops in the presence of bound enzyme. The results were interpreted spectrophotometrically. According to the manufacturer’s recommendations 5, for a dual wavelength reading (450-630 nm), a cut-off value of 0.130 Optical Density (OD) was adopted. Statistical analysis HpSA test sensitivity, specificity and their 95% confidence intervals (CI), positive and negative predictive values were calculated according to standard methods, assuming 13C-UBT as reference test. The rates of discrepancy and the mean HpSA ODs obtained in treated and untreated subjects were compared using chi-square and Student’s t tests. p
Results Overall UBT was positive in 40 cases and negative in 85, while HpSA was positive in 71 cases and negative in 54. Conflicting results were, therefore, found in
TableI. Urea breath test (UBTI and /-ie/h#wter
pyhri in stool specimens tHpSA1. Rae&e in treated and urwaated subjects.
edt@?&e f@l * UBT+
19
llrarc 0
UBT-
10
23
~a, w* 18 24
lw3 28
?%a Il. Sensitivity, specificity, predictive positive end negative values of HpSA vs UBT in Wee&d and untreated subjects EW% confidence limits txport%d in brackatsl.
URageted subjects
Treated subjects
I
SP
100%
70%
(83.lOO%l
[52-85%)
86%
54%
[64-EMd
(40-66%1
pII+
PM-
66%
I 00%
44%
91%
30% of subjects (37/125); 34 out of 37 cases presented a positive HpSA test in the presence of negative UBT. In detail, in untreated subjects the discrepancy rate was 19% (10/52) and was due, in all cases, to a positive HpSA associated with a negative UBT. In treated subjects, conflicting results were detected in 27 cases: 24 negative UBT with positive HpSA and 3 positive UBT with negative HpSA (discrepancy rate 37%). These results are outlined in Table I. Table II shows HpSA sensitivity, specifitiy, positive and negative predictive values, for treated and untreated subjects. As far as concerns subjects after treatment, the discrepancies did not correlate with the time elapsing between the end of treatment and the test itself, although a trend towards a greater discrepancy was observed in patients evaluated within 6 months of therapy. HpSA ODs measured in treated and untreated subjects vs H. pylon! status as defined by UBT are shown in Figure 1. Mean HpSA OD in subjects with positive results of both UBT and HpSA was 1.192 (range 0.1752.500). In patients with conflicting results of the two tests (positive HpSA and negative UBT), mean HpSA OD and mean UBT DOB-30 were, respectively, 0.190 (range 0.155- 0.297), 1.23 (range 0.5-2.0) in untreated subjects and 0.286 (range: 0.140-1020), 1.58 (range 0.3-3.1) in treated patients. The comparison between treated and untreated sub-
jects indicates that both the percentage of conflicting results (37% vs 19%) and their corresponding mean HpSA OD values (0.286 vs 0.190) are significantly different in the two groups (pcO.005). HpSA inefflciency vs HpSA cut-off value is shown in Figure 2 (panel a: untreated, panel b: treated). The inefficiency range variability (error band) calculated by the Bootstrap analysis at 95% CI is also shown in the same Figure (feint line). In untreated subjects, it clearly appears as raising the cut off value above 0.200 OD, the discrepancy falls below the significance level (zero value of inefficiency included in the error band): this means that, in such conditions, UBT and HpSA can be considered equivalent in assessing H. pylori status. In
Fig. 1. HpsA qdcal &ns@ datarminathr& in tre@ad Lt.1 and un. treated sub&lz lo). The vahea are p&&&d, against IJBT me&a. Up per normal MpSA limit (dot&d line1 is indic;ated.
287
Discrepancy
behwen
--
HpSA and UBT
0
0.4OQ
RZOO
0.600
0.800
1.000
80 80 70 860 so 40 30 20 10 I
1
--f
l~2-q
0
1
80 70 * 60 30 40 30 20 IO 0 0
0.200
0.400
0.600
0.800
1.ooo
B$ahmwftvak
Vg. Z. HpsA inefficiency &hick ~urval calculated at different cut off ralwes in treated and ur%raated patients (upper and lower panel). Er‘or band &$~t line1 was inferred by Bootstrap method. An arrow inketes the manufecturers cut off value.
treated patients (panel b), this can not be achieved, even raising the cut-off value, implying that additional non-methodological mechanisms are involved in HpSA inefficiency.
Discussion Among the non-invasive methods, UBT is generally accepted as the gold standard for the diagnosis of H. pylori infection. The test has been reported to be even more accurate and reproducible than histology and its use has been particularly recommended for the evaluation of treatment success, when endoscopic reassessment is not needed4. The test is based on the urease 288
activity of the bacterium and it is a measure of current infection. The Premier Platinum HpSA enzyme immunoassay is a qualitative procedure for the detection of H. pylori antigens in human stools and has been claimed to be specific for H. pylori, showing no cross reactivity with any of the many other organisms potentially present in the faeces 5. At variance with previous reports *-‘O,in our experience, the agreement between 13C-UBT and HpSA proved to be quite unsatisfactory: 70% in the population under study. Examining our data it emerges that: - The discrepancy is mostly found in the group of “treated” patients (37% vs 19%). - With the exception of 3 cases, the discordance is always due to positivity of HpSA associated with negative UBT. Similar results were also found by Trevisani et al. “, who showed that, in treated subjects, HpSA positivity associated with negative UBT can be found up to six months after the end of treatment. Interestingly, in our experience, mean HpSA value, in such cases, was far lower than in subjects in whom both tests were positive (0.273 vs 1.192 OD). - In untreated patients, the HpSA inefficiency curve shows cut off values at which the inefficiency is negligible, i.e., HpSA can be considered equivalent to UBT in assessing H. pylori status; in patients tested after eradication treatment this never occurs. On the grounds of these data, it seems reasonable to postulate that the suggested HpSA upper normal limit is too low and this might explain the discrepancy detected in untreated subjects, while in treated patients additional factors must be involved. In this regard, it is important to bear in mind the different target of the two assays: measurement of enzymatic activity for UBT ‘* and immunological identification of H. pylori antigens for HpSA. It is well known that H. pylori can exist in two forms: spiral and coccoid-like I3 I4 and that the latter can be induced by the administration of antibiotics or antisecretory drugs, mainly proton pump inhibitors ‘s-‘7. In addition, coccoid forms bind poorly to gastric epithelial cells but can be easily found in stools lg. If we consider the loss of urease activity I5 I’) but not of the immunogenic properties of coccoid bacteria *O2’, we might speculate that some of the detected discrepancies in treated subjects are due to the identification of coccoid forms only by the immunoassay but not by UBT. The significantly higher percentage of conflicting results observed between UBT and HpSA in treated patients, especially in those evaluated within 6 months of treatment, might support this hypothesis. Similar findings of persistence of H. pylori antigens, detected both with polymerase chain reaction (PCR) and enzyme immuno-assay (EIA), in the stool of successfully eradicated patients have been
G. Maraerc
reported by Makristathis et al. 22 Furthermore, in an animal model, Cellini et al. 23, demonstrated that, up to 3 months after inoculation, “viable but not culturable” H. pylori forms can still be detected in the mouse stomach. It should be noted that previous reports of good agreement between the two tests are based mainly on results obtained in untreated subjects; data concerning post-treatment evaluation are still scanty and contradictory and do not allow any definite conclusions to be made on HpSA and UBT agreement in this population 9-‘1. Furthermore, UBT negative results in the presence of coccoid forms in the gastric antrum have already been reported by others comparing ureasebased tests vs histology 24. A larger prospective study comparing UBT, HpSA and histology plus immunostaining on gastric biopsies is currently in progress in our laboratory to verify this hypothesis, which can have significant implications from a clinical and epidemiological point of view. The knowledge that H. pylori can revert to a coccoid form has in fact stimulated speculation about its role in transmission 2s and as a possible cause of reinfection in duodenal ulcer disease, although the potential of such forms to regrowth into spiral, hence virulent, forms is still a matter of debate 26-29. Although the presence of coccoid forms in the stomach may play a role in a proportion of discrepant tests, we are aware that this can not be the only explanation of our findings. Additional mechanisms should, therefore, be considered in the interpretation of these intriguing results, such as: 1. underreporting of drug intake interfering with UBT in spite of our careful interview of the patients on this topic; by HpSA of Helicobacter of intesti2. identification nal origin other than H. pylori species. 3. persistence in the intestinal tract of H. pylori as response to the eradication treatment. Up to now, we have no way to confirm or reject any of these hypotheses. In conclusion, according to our results, while a negative HpSA test correlates well with UBT, a certain degree of discrepancy can be appreciated in the presence of positive results, mainly in subjects evaluated after eradication treatment. Whether these contradictory results are due to methodological pitfalls or to the different background of the two tests still needs to be ascertained. Immunostaining on histological specimens together with repeated UBT during the follow-up could be considered in post-treatment patients with negative UBT and positive HpSA. H. pylori detection in stool by immunoassay might prove to be a useful tool in the understanding of H. pylori infection transmission and peptic ulcer relapse.
et al.
References ’ Agreus L, Talley N. Challenges in managing al practice. Br Med J 1997;315:1284-8. ? Cutler AF, Havstad bert IT. Accuracy Helicobacter pylori 1 Steen T, Berstad 14C urea breath 1996;l lO:A102.
dyspepsia
in gener-
S, Ma CK, Blaser MJ, Perez-Perez GI, Schuof invasive and noninvasive tests to diagnose infection. Gastroenterology 199.5; 109: 136-4 1.
K, Meling test repeated
T, Berstad A. Reproducibility of the after one week. Am J Gastroenterol
4 Atherton JC. Non-endoscopic tests in the diagnosis of Helicobacter pylori infection. Aliment Pharmacol Ther 1997; 11 (Suppl. 1): 1 l-20. 5 Kozak K, Larka C, Nick01 A Yi A. Detection of Helicobacter pylori antigen in stool specimens using a novel enzyme immunoassay (abstract). American Society for Microbiology, May 4-6, 1997, Miami (USA):C271. h Logan RPH, Dill S, E&hart Bauer F, Walker MM, Hierschl AM, Gummett PA, et al. The European 13Curea breath test for the detection of Helicobacter pylori. Eur J Gastroenterol Hepatol 1991;3:905-11. 7 Efron B. The jackknife, Philadelphia: Society 1992.
the bootstrap for Industrial
and other resampling plans. and Applied Mathematics;
* Vaira D, Menegatti M, Acciardi C, Landi F. Ricci C, Ali A, et al. A new “antigen” assay on stool specimen for Helicobacter pylori. Preliminary report. Digestion 1998;59(Suppl 3):477. 9 Vaira D, Malfertheiner P, Megraud F, Axon A, Deltenre M, Dixon M, et al. A new “antigen” assay on stool specimen (SS) for Helicobacter pylori (HP). European multicentre study. Ital J Gastroenterol Hepatol 1998;3O(Suppl 2):A90. ‘” Widmayer C, Rorai E, Peri A, Urban F, Vram A, Buri L, et al. Clinical validation of HpSa immunoassay for an Helicobacter pylori “antigen” in stools. Ital J Gastroenterol Hepatol 1998;3O(Suppl 2):A123. ” Trevisani L, Sartori S, Galvani F, Rossi MR, Ruina M, Chiamenti C, et al. Evaluation of a new enzyme immunoassay for detecting Helicobacter pylori in feces: a prospective pilot study. Am J Gastroenterol 1999;94: 1830-3. ‘* Logan RPH. The “C-UBT for the detection of Helicobacter pylori. In: Rathbone BJ, Heatley RV, editors. Helicobacter pylori and gastroduodenal disease (2”” ed). London: Blackwell; 1992. p. 88-107. I3 Goodwin CS, Armstrong JA. Microbiological aspects of Helicobacter pylori (Campylobacter pylori). Eur J Clin Microbial Infect Dis 1990;9:1-13. I4 Janas B, Czkwianianc E, Bak-Romaniszyn L, Bartel H, Tosik MD, Planeta-Malecka I. Electron microscopic study of association between coccoid forms of Helicobacter pylori and gastric epithelial cells. Am J Castroenterol 1995;90:1829-33. I5 Nilius M Strole A, Bode G, Malfertheiner P. Coccoid like forms (CLF) of’ Helicobacter pylori. Enzyme activity and antigenicity. Int J Med Microbial Virol Parasitol Infect Dis 1993;280:259-72. I6 Cellini L, Allocati lori: a fickle germ. ” Bode G, Mauch cobacter pylori. 1993; 111:483-90.
N, Angelucci D, Dainelli B. Helicobacter Microbial Immunol 1994;38:25-30. F, Malfetheiner Criteria for
P. The coccoid forms their viability. Epidemiol
I8 Cole SP, Cirillo D, Kagnoff MF, Guiney coid and spiral Helicobacter pylori differ here to gastric epithelial cells and induce Infect Immun 1997;65:843-6.
pyof HeliInfect
DG, Eckmann L. Cocin their abilities to adinterleukin-8 secretion.
I9 Narikawa S, Kawai S, Aoshima H, Kawamata 0, Hikiji K, et al. Comparison of the nucleic acids coccoid forms of Helicobacter pylori. Clin Diagn 1997;4:285-90.
Kawaguchi R, of helical and Lab Immunol
289
Discrepancy
between HpSA and UBT
*’ Cao J, Li ZQ, Borch K, Petersson F, Mardh S. Comparison of the nuclei acids of helical and coccoid forms of Helicobacter pylori using a murine monoclonal antibody. Clin Chim Acta 1997;267: 183-96. ” Vijayakumari S, Khin MM, Jiang B, Ho B. The pathogenic role of the coccoid form of Helicobacer pylori. Cytobios 1995;82:251-60. ” Makristathis A, Paschin E, Schutze K, Wimmer M, Rotter ML, Hirsch1 AM. Detection of Helicobacter pylori in stool specimens by PCR and antigen enzyme immunoassay. J Clin Microbial 1998;36:2772-4. 23 Cellini L, Dainelli B, Angelucci D, Grossi L, Di Bartolomeo S, Di Campli E, et al. Evidence for an oral-faecal transmission of Helicobacter pylori infection in an experimental murine model. APMIS 1999;107:477-84. ?4 Neri M. Coccoid forms of H. pylori are a cause of falsely nega-
tive urease-based tests. Ital J Gastroenterol Hepatol 19983O(Suppl 2):A115. x Goodman KJ, Correa P. The transmission of Helicobacter pylori. A critical review of the evidence. Int J Epidemiol 1995;24:875-87. 26 Goodwin CS, Worsley BW. Microbiology of Helicobacter pylori. Gastroenterol Clin North Am 1993;22:5-19. 27 Cellini L, Allocati N, Angelucci D, Iezzi T, Di Campli E, Marzio L, et al. Coccoid Helicobacter pylori not culturable in vitro reverts in mice. Microbial Immunol 1994;38:843-50. 28 Eaton KA, Catrenich CE, Makin KM, Krakowka S. Virulence of coccoid forms of Helicobacter pylori on gnotobiotic piglets. J Infect Dis 1995;171:459-62. 29 Hua J, Megraud F. Evidence of the viability of non culturable coccoidal forms of Helicobacter felis. Gut 1995;37(Suppl l):A94.
DIGEST LIVER OIS 2000;32:285-90
Discrepancy between Helicobacter pyiori stool antigen assay and urea breath test in the detection of Helicobacter pylori infection G. L. P. S. C. A. A.
Masoero Lombard0 Della Monica’ Vicari Crocilk2 Duglio2 Pera
Background. The reference diagnostic methods available for detection of Helicobacter pylori infection are either invasive [histology] or expensive and highly sophisticated [Urea Breath Test]. A new enzyme immunoassay, which can be easily performed in any laboratory has been developed to detect Helicobacter pylori in stool specimens [HpSA - Meridian Diagnostics, Cincinnati, USA]. Aim of the study was to compare HpSA to Urea Breath Test. Patients and methods. A total of 125 patients (52 never treated for Helicobacter pylori infection and 73 after Helicobacter pylori eradication therapy] referring to our Department, underwent both tests within two weeks. Results. Contrasting results between the two tests were found in 30% of cases: in 19% of the untreated patients and in 37% of the treated patients [p
Digest
Liver
Key words: test
Dis 2000;32:295-90 coccoid
forms
of
Helicobacter py/wi;
Helicobacter py/ori
stool
assay;
urea
breath
The best technique for the diagnosis of Helicobacter pylori (H. pylori) infection is yet to be identified, and a universally accepted gold standard is still lacking. Diagnostic methods may be invasive (endoscopic) or non-invasive (non-endoscopic). Among the former, histological examination of multiple biopsy specimens is accepted as reference method, although it includes disadvantages related to all biopsy methods, due to sampling errors and observers’ variations. Among the non-invasive methods, the 13C -Urea
Discrepancy
between HpSl and UBT
Breath test (UBT) is considered the most reliable in assessing H. pylori infection both before and after eradication ‘; when carried out at least four weeks after the end of treatment, it would appear to be as accurate as biopsy-based tests, perhaps even more * 3. Serological tests vary greatly in accuracy and appear to be less reliable than UBT, especially in the post treatment assessment 4. However, since the UBT-technique requires highly sophisticated and expensive equipment along with specialised staff - not universally available - the demand for a simpler, equally reliable and cost-effective test for H. pylori detection is persistently high. Recently, a new enzyme immunoassay test 5 has been developed to detect H. pylori in stool specimens, namely Meridian Diagnostics, HpSA, Cincinnati, USA; this test has the advantage that it is easy to perform in any laboratory thanks to the possibility of the simple visual or spectrophotometric interpretation. Aim of our study was to compare HpSA to 13C-UBT in a group of patients referring to our GI unit. UBT was adopted as gold standard.
Patients and methods Patients A total of 125 consecutive patients, either dyspeptic or with a previous history of peptic disease (68 females and 57 males; mean age 52 years, range 15-79) referring to our GI Unit for H. pylori assessment by means of UBT, were included in the study. Of these, 52 subjects had never been submitted to any H. pylori eradication therapy; in 73 cases, the indication for UBT was to evaluate success of treatment. The time elapsing between the end of treatment and the test itself ranged from 2 to 212 months. Exclusion criteria were: use of antisecretory drugs, bismuth-based compounds or antibiotics within 2 months prior to the two tests. Fresh stool specimens were collected within 1 week of UBT and immediately stored at -20°C until the running of the assay. Methods Urea breath test The test was performed according to the European protocol 6. Fasting subjects were given a standard meal (200 ml of full cream milk or orange juice) to delay gastric emptying, followed by a solution of 100 mg of urea isotopically labelled with 13C. Duplicate breath samples were collected in basal conditions and 30 minutes after ingestion of 13C-Urea. The 13C enrichment of samples was determined by isotope ratio mass spectrometry (ABCA- Europa Scientific, Crewe, UK). A test was considered positive when the difference between the ‘“C-enrichment at 30 286
minutes over the baseline (DOB-30) was >4 per mil. In our laboratory, where UBT has been routinely performed for 3 years, the sensitivity (SS) and specificity (SP) of the test, evaluated using histology as gold standard, are 92% and 97%, respectively. H. pylori stool assay Faecal H. pylori detection was performed by means of a commercially available kit (Premier Platinum HpSA - Meridian Diagnostics, Cincinnati, USA). The test uses polyclonal anti-H. pyZori antibodies adsorbed to microwells. Diluted stool samples and a peroxidase conjugated polyclonal antibody are added to the wells and incubated for 1 h at room temperature. Substrate is added and incubated for 10 minutes. Colour develops in the presence of bound enzyme. The results were interpreted spectrophotometrically. According to the manufacturer’s recommendations 5, for a dual wavelength reading (450-630 nm), a cut-off value of 0.130 Optical Density (OD) was adopted. Statistical analysis HpSA test sensitivity, specificity and their 95% confidence intervals (CI), positive and negative predictive values were calculated according to standard methods, assuming 13C-UBT as reference test. The rates of discrepancy and the mean HpSA ODs obtained in treated and untreated subjects were compared using chi-square and Student’s t tests. p
Results Overall UBT was positive in 40 cases and negative in 85, while HpSA was positive in 71 cases and negative in 54. Conflicting results were, therefore, found in
TableI. Urea breath test (UBTI and /-ie/h#wter
pyhri in stool specimens tHpSA1. Rae&e in treated and urwaated subjects.
edt@?&e f@l * UBT+
19
llrarc 0
UBT-
10
23
~a, w* 18 24
lw3 28
?%a Il. Sensitivity, specificity, predictive positive end negative values of HpSA vs UBT in Wee&d and untreated subjects EW% confidence limits txport%d in brackatsl.
URageted subjects
Treated subjects
I
SP
100%
70%
(83.lOO%l
[52-85%)
86%
54%
[64-EMd
(40-66%1
pII+
PM-
66%
I 00%
44%
91%
30% of subjects (37/125); 34 out of 37 cases presented a positive HpSA test in the presence of negative UBT. In detail, in untreated subjects the discrepancy rate was 19% (10/52) and was due, in all cases, to a positive HpSA associated with a negative UBT. In treated subjects, conflicting results were detected in 27 cases: 24 negative UBT with positive HpSA and 3 positive UBT with negative HpSA (discrepancy rate 37%). These results are outlined in Table I. Table II shows HpSA sensitivity, specifitiy, positive and negative predictive values, for treated and untreated subjects. As far as concerns subjects after treatment, the discrepancies did not correlate with the time elapsing between the end of treatment and the test itself, although a trend towards a greater discrepancy was observed in patients evaluated within 6 months of therapy. HpSA ODs measured in treated and untreated subjects vs H. pylon! status as defined by UBT are shown in Figure 1. Mean HpSA OD in subjects with positive results of both UBT and HpSA was 1.192 (range 0.1752.500). In patients with conflicting results of the two tests (positive HpSA and negative UBT), mean HpSA OD and mean UBT DOB-30 were, respectively, 0.190 (range 0.155- 0.297), 1.23 (range 0.5-2.0) in untreated subjects and 0.286 (range: 0.140-1020), 1.58 (range 0.3-3.1) in treated patients. The comparison between treated and untreated sub-
jects indicates that both the percentage of conflicting results (37% vs 19%) and their corresponding mean HpSA OD values (0.286 vs 0.190) are significantly different in the two groups (pcO.005). HpSA inefflciency vs HpSA cut-off value is shown in Figure 2 (panel a: untreated, panel b: treated). The inefficiency range variability (error band) calculated by the Bootstrap analysis at 95% CI is also shown in the same Figure (feint line). In untreated subjects, it clearly appears as raising the cut off value above 0.200 OD, the discrepancy falls below the significance level (zero value of inefficiency included in the error band): this means that, in such conditions, UBT and HpSA can be considered equivalent in assessing H. pylori status. In
Fig. 1. HpsA qdcal &ns@ datarminathr& in tre@ad Lt.1 and un. treated sub&lz lo). The vahea are p&&&d, against IJBT me&a. Up per normal MpSA limit (dot&d line1 is indic;ated.
287
Discrepancy
behwen
--
HpSA and UBT
0
0.4OQ
RZOO
0.600
0.800
1.000
80 80 70 860 so 40 30 20 10 I
1
--f
l~2-q
0
1
80 70 * 60 30 40 30 20 IO 0 0
0.200
0.400
0.600
0.800
1.ooo
B$ahmwftvak
Vg. Z. HpsA inefficiency &hick ~urval calculated at different cut off ralwes in treated and ur%raated patients (upper and lower panel). Er‘or band &$~t line1 was inferred by Bootstrap method. An arrow inketes the manufecturers cut off value.
treated patients (panel b), this can not be achieved, even raising the cut-off value, implying that additional non-methodological mechanisms are involved in HpSA inefficiency.
Discussion Among the non-invasive methods, UBT is generally accepted as the gold standard for the diagnosis of H. pylori infection. The test has been reported to be even more accurate and reproducible than histology and its use has been particularly recommended for the evaluation of treatment success, when endoscopic reassessment is not needed4. The test is based on the urease 288
activity of the bacterium and it is a measure of current infection. The Premier Platinum HpSA enzyme immunoassay is a qualitative procedure for the detection of H. pylori antigens in human stools and has been claimed to be specific for H. pylori, showing no cross reactivity with any of the many other organisms potentially present in the faeces 5. At variance with previous reports *-‘O,in our experience, the agreement between 13C-UBT and HpSA proved to be quite unsatisfactory: 70% in the population under study. Examining our data it emerges that: - The discrepancy is mostly found in the group of “treated” patients (37% vs 19%). - With the exception of 3 cases, the discordance is always due to positivity of HpSA associated with negative UBT. Similar results were also found by Trevisani et al. “, who showed that, in treated subjects, HpSA positivity associated with negative UBT can be found up to six months after the end of treatment. Interestingly, in our experience, mean HpSA value, in such cases, was far lower than in subjects in whom both tests were positive (0.273 vs 1.192 OD). - In untreated patients, the HpSA inefficiency curve shows cut off values at which the inefficiency is negligible, i.e., HpSA can be considered equivalent to UBT in assessing H. pylori status; in patients tested after eradication treatment this never occurs. On the grounds of these data, it seems reasonable to postulate that the suggested HpSA upper normal limit is too low and this might explain the discrepancy detected in untreated subjects, while in treated patients additional factors must be involved. In this regard, it is important to bear in mind the different target of the two assays: measurement of enzymatic activity for UBT ‘* and immunological identification of H. pylori antigens for HpSA. It is well known that H. pylori can exist in two forms: spiral and coccoid-like I3 I4 and that the latter can be induced by the administration of antibiotics or antisecretory drugs, mainly proton pump inhibitors ‘s-‘7. In addition, coccoid forms bind poorly to gastric epithelial cells but can be easily found in stools lg. If we consider the loss of urease activity I5 I’) but not of the immunogenic properties of coccoid bacteria *O2’, we might speculate that some of the detected discrepancies in treated subjects are due to the identification of coccoid forms only by the immunoassay but not by UBT. The significantly higher percentage of conflicting results observed between UBT and HpSA in treated patients, especially in those evaluated within 6 months of treatment, might support this hypothesis. Similar findings of persistence of H. pylori antigens, detected both with polymerase chain reaction (PCR) and enzyme immuno-assay (EIA), in the stool of successfully eradicated patients have been
G. Maraerc
reported by Makristathis et al. 22 Furthermore, in an animal model, Cellini et al. 23, demonstrated that, up to 3 months after inoculation, “viable but not culturable” H. pylori forms can still be detected in the mouse stomach. It should be noted that previous reports of good agreement between the two tests are based mainly on results obtained in untreated subjects; data concerning post-treatment evaluation are still scanty and contradictory and do not allow any definite conclusions to be made on HpSA and UBT agreement in this population 9-‘1. Furthermore, UBT negative results in the presence of coccoid forms in the gastric antrum have already been reported by others comparing ureasebased tests vs histology 24. A larger prospective study comparing UBT, HpSA and histology plus immunostaining on gastric biopsies is currently in progress in our laboratory to verify this hypothesis, which can have significant implications from a clinical and epidemiological point of view. The knowledge that H. pylori can revert to a coccoid form has in fact stimulated speculation about its role in transmission 2s and as a possible cause of reinfection in duodenal ulcer disease, although the potential of such forms to regrowth into spiral, hence virulent, forms is still a matter of debate 26-29. Although the presence of coccoid forms in the stomach may play a role in a proportion of discrepant tests, we are aware that this can not be the only explanation of our findings. Additional mechanisms should, therefore, be considered in the interpretation of these intriguing results, such as: 1. underreporting of drug intake interfering with UBT in spite of our careful interview of the patients on this topic; by HpSA of Helicobacter of intesti2. identification nal origin other than H. pylori species. 3. persistence in the intestinal tract of H. pylori as response to the eradication treatment. Up to now, we have no way to confirm or reject any of these hypotheses. In conclusion, according to our results, while a negative HpSA test correlates well with UBT, a certain degree of discrepancy can be appreciated in the presence of positive results, mainly in subjects evaluated after eradication treatment. Whether these contradictory results are due to methodological pitfalls or to the different background of the two tests still needs to be ascertained. Immunostaining on histological specimens together with repeated UBT during the follow-up could be considered in post-treatment patients with negative UBT and positive HpSA. H. pylori detection in stool by immunoassay might prove to be a useful tool in the understanding of H. pylori infection transmission and peptic ulcer relapse.
et al.
References ’ Agreus L, Talley N. Challenges in managing al practice. Br Med J 1997;315:1284-8. ? Cutler AF, Havstad bert IT. Accuracy Helicobacter pylori 1 Steen T, Berstad 14C urea breath 1996;l lO:A102.
dyspepsia
in gener-
S, Ma CK, Blaser MJ, Perez-Perez GI, Schuof invasive and noninvasive tests to diagnose infection. Gastroenterology 199.5; 109: 136-4 1.
K, Meling test repeated
T, Berstad A. Reproducibility of the after one week. Am J Gastroenterol
4 Atherton JC. Non-endoscopic tests in the diagnosis of Helicobacter pylori infection. Aliment Pharmacol Ther 1997; 11 (Suppl. 1): 1 l-20. 5 Kozak K, Larka C, Nick01 A Yi A. Detection of Helicobacter pylori antigen in stool specimens using a novel enzyme immunoassay (abstract). American Society for Microbiology, May 4-6, 1997, Miami (USA):C271. h Logan RPH, Dill S, E&hart Bauer F, Walker MM, Hierschl AM, Gummett PA, et al. The European 13Curea breath test for the detection of Helicobacter pylori. Eur J Gastroenterol Hepatol 1991;3:905-11. 7 Efron B. The jackknife, Philadelphia: Society 1992.
the bootstrap for Industrial
and other resampling plans. and Applied Mathematics;
* Vaira D, Menegatti M, Acciardi C, Landi F. Ricci C, Ali A, et al. A new “antigen” assay on stool specimen for Helicobacter pylori. Preliminary report. Digestion 1998;59(Suppl 3):477. 9 Vaira D, Malfertheiner P, Megraud F, Axon A, Deltenre M, Dixon M, et al. A new “antigen” assay on stool specimen (SS) for Helicobacter pylori (HP). European multicentre study. Ital J Gastroenterol Hepatol 1998;3O(Suppl 2):A90. ‘” Widmayer C, Rorai E, Peri A, Urban F, Vram A, Buri L, et al. Clinical validation of HpSa immunoassay for an Helicobacter pylori “antigen” in stools. Ital J Gastroenterol Hepatol 1998;3O(Suppl 2):A123. ” Trevisani L, Sartori S, Galvani F, Rossi MR, Ruina M, Chiamenti C, et al. Evaluation of a new enzyme immunoassay for detecting Helicobacter pylori in feces: a prospective pilot study. Am J Gastroenterol 1999;94: 1830-3. ‘* Logan RPH. The “C-UBT for the detection of Helicobacter pylori. In: Rathbone BJ, Heatley RV, editors. Helicobacter pylori and gastroduodenal disease (2”” ed). London: Blackwell; 1992. p. 88-107. I3 Goodwin CS, Armstrong JA. Microbiological aspects of Helicobacter pylori (Campylobacter pylori). Eur J Clin Microbial Infect Dis 1990;9:1-13. I4 Janas B, Czkwianianc E, Bak-Romaniszyn L, Bartel H, Tosik MD, Planeta-Malecka I. Electron microscopic study of association between coccoid forms of Helicobacter pylori and gastric epithelial cells. Am J Castroenterol 1995;90:1829-33. I5 Nilius M Strole A, Bode G, Malfertheiner P. Coccoid like forms (CLF) of’ Helicobacter pylori. Enzyme activity and antigenicity. Int J Med Microbial Virol Parasitol Infect Dis 1993;280:259-72. I6 Cellini L, Allocati lori: a fickle germ. ” Bode G, Mauch cobacter pylori. 1993; 111:483-90.
N, Angelucci D, Dainelli B. Helicobacter Microbial Immunol 1994;38:25-30. F, Malfetheiner Criteria for
P. The coccoid forms their viability. Epidemiol
I8 Cole SP, Cirillo D, Kagnoff MF, Guiney coid and spiral Helicobacter pylori differ here to gastric epithelial cells and induce Infect Immun 1997;65:843-6.
pyof HeliInfect
DG, Eckmann L. Cocin their abilities to adinterleukin-8 secretion.
I9 Narikawa S, Kawai S, Aoshima H, Kawamata 0, Hikiji K, et al. Comparison of the nucleic acids coccoid forms of Helicobacter pylori. Clin Diagn 1997;4:285-90.
Kawaguchi R, of helical and Lab Immunol
289
Discrepancy
between HpSA and UBT
*’ Cao J, Li ZQ, Borch K, Petersson F, Mardh S. Comparison of the nuclei acids of helical and coccoid forms of Helicobacter pylori using a murine monoclonal antibody. Clin Chim Acta 1997;267: 183-96. ” Vijayakumari S, Khin MM, Jiang B, Ho B. The pathogenic role of the coccoid form of Helicobacer pylori. Cytobios 1995;82:251-60. ” Makristathis A, Paschin E, Schutze K, Wimmer M, Rotter ML, Hirsch1 AM. Detection of Helicobacter pylori in stool specimens by PCR and antigen enzyme immunoassay. J Clin Microbial 1998;36:2772-4. 23 Cellini L, Dainelli B, Angelucci D, Grossi L, Di Bartolomeo S, Di Campli E, et al. Evidence for an oral-faecal transmission of Helicobacter pylori infection in an experimental murine model. APMIS 1999;107:477-84. ?4 Neri M. Coccoid forms of H. pylori are a cause of falsely nega-
tive urease-based tests. Ital J Gastroenterol Hepatol 19983O(Suppl 2):A115. x Goodman KJ, Correa P. The transmission of Helicobacter pylori. A critical review of the evidence. Int J Epidemiol 1995;24:875-87. 26 Goodwin CS, Worsley BW. Microbiology of Helicobacter pylori. Gastroenterol Clin North Am 1993;22:5-19. 27 Cellini L, Allocati N, Angelucci D, Iezzi T, Di Campli E, Marzio L, et al. Coccoid Helicobacter pylori not culturable in vitro reverts in mice. Microbial Immunol 1994;38:843-50. 28 Eaton KA, Catrenich CE, Makin KM, Krakowka S. Virulence of coccoid forms of Helicobacter pylori on gnotobiotic piglets. J Infect Dis 1995;171:459-62. 29 Hua J, Megraud F. Evidence of the viability of non culturable coccoidal forms of Helicobacter felis. Gut 1995;37(Suppl l):A94.