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Evaluation of a latex agglutination test (PYLOGEN) for the detection of Helicobacter pylori in stool specimens
Available online at www.sciencedirect.com
Diagnostic Microbiology and Infectious Disease 63 (2009) 349 – 353 www.elsevier.com/locate/diagmicrobio
Bacteriology
Evaluation of a latex agglutination test (PYLOGEN) for the detection of Helicobacter pylori in stool specimens Silvia Blancoa,b , Montse Fornéc , Alicia Lacomaa,b , Cristina Prata,b , Miguel Ángel Cuestaa , Loreto Fuenzalidaa,b , Josep Maria Viverc , Jose Domíngueza,b,⁎ a
Servei de Microbiologia, Hospital Universitari Germans Trias i Pujol, Fundació Institut d'Investigació Germans Trias i Pujol, Badalona, 08916 Barcelona, Spain b Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain c Servei de Gastroenterologia, Hospital Universitari Mútua de Terrassa, 08221 Barcelona, Spain Received 17 October 2008; accepted 15 December 2008
Abstract The aim of the study was to assess a new latex agglutination (LA) stool antigen assay (PYLOGEN; CerTest Biotec, Zaragoza, Spain) in the diagnosis of Helicobacter pylori infection and to monitor its eradication after treatment. The LA test has been approved for sale in Europe, and its approval from the US Food and Drug Administration is still pending. The individuals enrolled were classified into 3 groups of patients: Group 1 consisted of 38 patients who are H. pylori positive. The diagnosis of H. pylori infection was established if there was concordance between 2 test results (urea breath test [UBT], rapid urease test, and histopathologic study) or if the culture alone was positive. Patients with only 1 positive test were considered indeterminate and were excluded from the study. Group 2 comprised 9 patients without positive tests and who were considered to be H. pylori negative. Group 3 consisted of 57 patients who received eradication treatment. The sensitivity and specificity of the test were 78.9% and 100%, respectively. The results of the UBT of the patients were studied 6 weeks after eradication therapy. The sensitivity and specificity of the LA test relative to UBT for patients after treatment were 75% and 93.3%, respectively. © 2009 Elsevier Inc. All rights reserved. Keywords: Helicobacter pylori; Stool antigen detection; Latex agglutination test; Monitoring eradication
1. Introduction Helicobacter pylori is recognized as one of the most common and medically important pathogens worldwide (Aksoy et al., 2003; Vaira et al., 2002a). In addition, it has been described as an etiologic agent in chronic gastritis and peptic ulcer disease and has also been associated with gastric malignancies (Asaka et al., 1994). Different invasive and noninvasive tests (Veijola et al., 2005b) are available for the diagnosis of H. pylori infection.
⁎ Corresponding author. Servei de Microbiologia, Fundació Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol, Ctra. del Canyet s/n, Badalona, 08916 Barcelona, Spain. Tel.: +34-934978894; fax: +34-934978895. E-mail address: [email protected] (J. Domínguez). 0732-8893/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2008.12.006
Although detection of H. pylori antigen in stool samples has been a subject of controversy, especially because of its poor correlation with the urea breath test (UBT) in the confirmation of eradication of H. pylori, it has been included in several clinical guidelines. Nowadays, stool antigen assays offer an alternative noninvasive method for the diagnosis of infection (Andrews et al., 2003; Forne et al., 2000; Islam et al., 2005; Vaira et al., 1999; Veijola et al., 2005a). Since the introduction of antigen stool assays, many studies have described the use of enzyme immunoassay (EIA) and immunochromatographic tests (ICTs) based on polyclonal or monoclonal antibodies for the diagnosis of H. pylori infections (Aksoy et al., 2003; Andrews et al., 2003; Blanco et al., 2008; Chisholm et al., 2004; Forne et al., 2000). Recently, the 1st latex agglutination (LA) assay for H. pylori antigen detection in stool samples has been developed. The aim of this study was to evaluate the
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usefulness of a new monoclonal antibody-based LA stool antigen assay (PYLOGEN; CerTest Biotec, Zaragoza, Spain) in the diagnosis of H. pylori infection and to monitor H. pylori eradication after treatment, comparing its accuracy with an ICT based on monoclonal antibody stool antigen assay (Letitest H. pylori CARD; Laboratorios Leti, Barcelona, Spain) and with 2 EIAs, one of them based on polyclonal antibodies (Premier Platinum HpSA; Meridian Diagnostics, Cincinnati, OH) and the other one based on monoclonal antibodies (Amplified IDEIA HpStAR; DakoCytomation, Cambridge, UK). 2. Methods 2.1. Patients The study population consisted of 104 patients reporting for routine upper gastrointestinal endoscopy at Hospital Universitari Mútua de Terrassa, Barcelona, Spain. Patients were selected retrospectively. Exclusion criteria were the recent use of bismuth-containing compounds or antibiotics (in the previous 2 months) or proton pump inhibitors (in the previous 15 days). The main diagnoses of the patients included ulcer (55.2%), antritis (18.4%), duodenitis (5.3%), and hiatal hernia (2.6%). UBT, rapid urease test (RUT), histopathologic study (H), culture in an antral biopsy specimen (Matrix test; Rohm Farm Laboratories, Germany), and detection of Helicobacter antigen by HpSA EIA test and HpStAR EIA were determined for all patients at the time initial diagnostic testing was performed. The diagnosis of H. pylori infection was established if there was concordance between 2 test results (RUT, UBT, or H) or if the culture alone was positive. Patients with only 1 positive test were considered indeterminate and were excluded from the study. We included patients during 2 stages of the treatment, at the time initial diagnostic testing was performed and 6 weeks after the patients finished the treatment, to evaluate the utility in assessing the eradication of the H. pylori. Individuals enrolled were classified into 3 groups: Group 1 consisted of 38 patients (14 female, mean age of 47.9 years) who are H. pylori positive (study group). Twelve patients had positive results for all tests (RUT, UBT, H, and culture), 18 patients had both results for RUT and H positive, and 8 patients had both results for RUT and UBT positive. Group 2 comprised 9 patients (3 female, mean age of 52.2 years) without any positive test (RUT, UBT, H, culture) who were considered to be H. pylori negative (control group). Group 3 consisted of 57 patients who received eradication treatment (2× omeprazole 20 mg, 2× clarithromycin 500 mg, and 2× amoxicillin 1 g for 7 days). The UBT results of the patients were studied after 6 weeks, and a stool sample was also collected to determine the presence of H. pylori antigen. In the posttreatment follow-up, UBT was used as the gold standard for considering eradication.
All samples were frozen at −70 °C before conducting the LA test and ICT; HpSA and HpStAR EIA tests were done before freezing and were not repeated. The study was approved by the ethics committees of the hospitals participating in the study. Patients expressed their consent to be included in the study. 2.2. Urea breath test UBT was performed in accordance with the European protocol (Dominguez-Munoz et al., 1997). Briefly, patients fasted overnight, and then a solution containing 4 g of citric acid was given before collecting baseline breath samples. Afterward, 100 mg of 13C-labeled urea dissolved in water was administered, and after 30 min, 2 breath samples were obtained. The increase in the mole fraction of tracer 13CO2 at 30 min compared with the baseline was expressed as atom percentage excess units. Of the results, more than 5% were considered positive, between 4.5% and 5% as indeterminate, and b4.5% as negative. 2.3. Stool antigen tests All the tests were performed, and the results were interpreted following the manufacturers' instructions. 2.4. PYLOGEN (CerTest Biotec) The latex reagent is a suspension of polystyrene latex particles of uniform size coated with monoclonal antibodies directed specifically against H. pylori antigen. The manufacture claims that the target antigen is of polysaccharide nature. Briefly, 100 μL of the latex reagent was mixed with 100 μL of diluted a stool sample on the slide. The results were read after a rotation of the slide for 10 min. The result was considered positive when the uniform appearance of the latex suspension converted to a clear agglutination. The LA test has been approved for sale in Europe, and its approval from the US Food and Drug Administration is still pending. 2.5. Letitest H. pylori CARD (Laboratorios Leti) The assay is an ICT based on monoclonal antibodies specific to H. pylori antigens. Five drops of a diluted sample were dispensed into the circular window marked with an arrow. The results were read after 10 min. If the result is negative, only 1 red band appears on the control line. If the sample is positive, a 2nd red band also appears in the sample line. 2.6. Premier Platinum HpSA EIA test (Meridian Diagnostics) The assay is based on polyclonal antibodies specific to H. pylori antigens. Diluted stool samples and a peroxidaseconjugated polyclonal antibody were added to the microwells. After washing the plate, substrate solution was added and incubated for 10 min at room temperature. The results were read by spectrophotometry. Absorbance (450/620 nm) ≥0.120 was considered positive.
S. Blanco et al. / Diagnostic Microbiology and Infectious Disease 63 (2009) 349–353
2.7. Amplified IDEIA™ HpStAR™ (DakoCytomation) The assay is an EIA based on monoclonal antibodies specific to H. pylori antigens. Diluted stool samples and a peroxidase-conjugated monoclonal antibody were added to the microwells and incubated for 1 h at room temperature. After washing the plate, the substrate solution was added and incubated for 10 min at room temperature. The results were read by spectrophotometric determination by means of an EIA microplate reader. An absorbance (450/620 nm) ≥0.150 was considered positive.
351
Table 2 Sensitivity and specificity of the stool antigen assays at the initial diagnosis of H. pylori infection and posttreatment follow-up Results antigen detection methods (%) LA Diagnosis Sensitivity 78.9 Specificity 100 Posttreatment follow-up Sensitivity 75 Specificity 93.3
Letitest ICT
HpSA EIA
HpStAR EIA
92.1 100
86.8 100
97.3 100
66.7 84.4
66.7 75.6
83.3 97.8
2.8. Statistical analysis Sensitivity and specificity were calculated in the usual manner. The agreement between different tests was estimated by Cohen's κ statistics. All analyses were made with SPSS statistical software for windows (SPSS version 15.0; SPSS, Chicago, IL). 3. Results Using LA, we detected H. pylori antigen in 30 of 38 samples from the study group (78.9% sensitivity) and in any of the patients from the control group (100% specificity). The sensitivity of Letitest ICT, HpStAR EIA, and HpSA EIA was 92.1%, 97.3%, and 86.8%, respectively. All the tests showed 100% specificity. In the 57 patients evaluated 6 weeks after the eradication therapy, the UBT result was negative in 45 patients, the LA test result was negative in 42 cases, the Letitest ICT result was negative in 38 cases, the HpSA EIA result was negative in 34, and the HpStAR EIA result was negative in 44 cases. The UBT result was positive for the remaining 12 patients, the LA test result was positive in 9 cases, the Letitest ICT result was positive in 8 cases, the HpSA EIA result was positive in 8 cases, and the HpStAR EIA result was positive in 10 cases. The overall agreements between the UBT and the antigen tests were as follows: LA, 89.4% (k = 0.683, SE =
Table 1 Results of the Helicobacter stool antigen methods in individuals infected and noninfected, at the time of diagnosis and after eradication therapy Results of the methods
LA Letitest ICT HpSA EIA HpStAR EIA
No. of individuals studied and their H. pylori status
Positive Negative Positive Negative Positive Negative Positive Negative
Before therapy
After therapy
Positive (n = 38)
Negative (n = 9)
Positive (n = 12)
Negative (n = 45)
30 8 35 3 33 5 37 1
0 9 0 9 0 9 0 9
9 3 8 4 8 4 10 2
3 42 7 38 9 34 1 44
0.120); Letitest ICT, 80.7% (k = 0.468; SE = 0.136); HpStAR EIA, 94.7% (k = 0.837; SE = 0.091); and HpSA EIA, 73.7% (k = 0.348; SE = 0.131). The sensitivity and specificity of LA test relative to UBT for patients after eradication therapy were 75% and 93.3%, respectively. The overall differences, including all group of patients, in the results between LA test and the Letitest ICT, HpSA EIA, and HpStAR EIA tests were not significant (P = 0.115, P = 0.089, P = 0.180, respectively). However, at the time initial diagnostic testing was performed, a significant difference only exists between the LA test and the HpStAR EIA test (P = 0.039). In the posttreatment follow-up, no significant differences between the antigen detection stool assays have been obtained. A summary of the results is shown in Tables 1 and 2.
4. Discussion The 1st antigen stool assay developed was the HpSA EIA assay, which uses polyclonal antibodies. This test has been extensively evaluated in pre- and posttreatment settings both in adults and children (Vaira et al., 2002b). It has been demonstrated that polyclonal assays are good tests for the diagnosis of infection but show low accuracy for monitoring eradication therapy (Aksoy et al., 2003; Forne et al., 2000; Islam et al., 2005; Ohkura et al., 2000; Vaira et al., 2002b). Previous evaluations comparing the polyclonal- and monoclonal-based assay results reveal comparable specificity but higher sensitivity of monoclonal tests for the diagnosis of H. pylori infection (Andrews et al., 2003; Asfeldt et al., 2004; Chisholm et al., 2004; Dominguez et al., 2006; Erzin et al., 2004; Weingart et al., 2004). As far as we know, this is the 1st time that an LA test has been evaluated for the diagnosis of H. pylori infection and eradication follow-up. This test has not been evaluated previously in comparison with other antigen tests. Our results show that the LA test is the less sensitive test among the tests evaluated for the initial diagnosis of H. pylori infection. On the contrary, among the antigen stool detection tests, the LA test and the HpStAR EIA are the more reliable methods, with the higher sensitivities and specificities, in monitoring the eradication after treatment. Nevertheless, in
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testing the eradication of the H. pylori at 6 weeks after finishing the treatment, any of the antigen stool detection tests evaluated had the same sensitivity as the UBT for determining persistent infection. The persistence of a positive antigen detection result in some patients with a negative UBT result 6 weeks after finishing the treatment has been explained by the longerlasting elimination of the low amounts of Helicobacter antigens from the degenerating remains of dead bacteria (Forne et al., 2000; Kusters et al., 1997). Therefore, the LA test, which had the lowest sensitivity at the diagnosis moment, has obtained better sensitivity and specificity than Letitest ICT and HpSA EIA in comparison with the UBT in patients who have eradicated the H. pylori. However, in our study, the test with a higher sensitivity (HpStAR) obtained the best agreement with the UBT. This fact could be explained by differences in the antigen detected. It has been hypothesized that HpStAR EIA may recognize a water-soluble antigen with a high turnover (Makristathis et al., 2000). The use of UBT alone may not be considered as the gold standard in the follow-up treatment. However, invasive diagnostic tests are not well tolerated by patients, and the value of UBT in assessing eradication of H. pylori has been clearly demonstrated. According to the Maastricht III Consensus Report (Malfertheiner et al., 2007), noninvasive tests should be used for confirmation of eradication, except in cases where repeat endoscopy is indicated, for example, patients with gastric ulcer. The main drawback of the current evaluation is that we studied a small group of patients. This fact could influence both the sensitivity and, specially, the specificity of the test evaluated. Another limitation is the lack of longitudinal follow-up in all the patients included at the moment of diagnosis. This was not possible because of logistical reasons. Despite these limitations, our study provides relevant data related to the clinical utility of the LA test for detection of H. pylori antigen in stool samples. Further studies including more patients could be adequate for confirming the results. LA assay is faster than the EIA, giving a result in a few minutes, and is easy to perform and interpret. LA test is comparable with ICT methods in technical complexity and the equipment required, allowing a wide diffusion of Helicobacter stool antigen detection in conventional laboratories, especially in areas with limited technical resources. Given that, some laboratories batch samples until they have enough to perform the EIA, causing delays in the diagnostic process. The LA test evaluated is especially useful for small hospital laboratories without appropriate equipment for performing the EIA and for laboratories that work with few samples. In conclusion, LA test is a method with a lower sensitivity than the other antigen assays tested, but with a high specificity to determine H. pylori infection. The test has 75% sensitivity and 93% specificity in comparison
with the UBT in monitoring Helicobacter eradication after therapy. Acknowledgments The authors would like to thank CerTest Biotec, Zaragoza, Spain, for providing the test kits required for the study. References Aksoy DY, et al. (2003) Evaluation of the Helicobacter pylori stool antigen test (HpSA) for the detection of Helicobacter pylori infection and comparison with other methods. Hepatogastroenterology 50: 1047–1049. Andrews J, et al. (2003) Comparison of three stool antigen tests for Helicobacter pylori detection. J Clin Pathol 56:769–771. Asaka M, et al. (1994) Possible role of Helicobacter pylori infection in early gastric cancer development. Cancer 73:2691–2694. Asfeldt A, et al. (2004) Accuracy of a monoclonal antibody-based stool antigen test in the diagnosis of Helicobacter pylori infection. Scand J Gastroenterol 39:1073–1077. Blanco S, et al. (2008) Comparison of stool antigen immunoassay methods for detecting Helicobacter pylori infection before and after eradication treatment. Diagn Microbiol Infect Dis 61:150–155. Chisholm SA, et al. (2004) Non-invasive diagnosis of Helicobacter pylori infection in adult dyspeptic patients by stool antigen detection: does the rapid immunochromatography test provide a reliable alternative to conventional ELISA kits? J Med Microbiol 53:623–627. Dominguez-Munoz JE, et al. (1997) A citric acid solution is an optimal test drink in the 13C-urea breath test for the diagnosis of Helicobacter pylori infection. Gut 40:459–462. Dominguez J, et al. (2006) Comparison of a monoclonal with a polyclonal antibody-based enzyme immunoassay stool test in diagnosing Helicobacter pylori infection before and after eradication therapy. Aliment Pharmacol Ther 23:1735–1740. Erzin Y, et al. (2004) Comparison of two different stool antigen tests for the primary diagnosis of Helicobacter pylori infection in Turkish patients with dyspepsia. Helicobacter 9:657–662. Forne M, et al. (2000) Accuracy of an enzyme immunoassay for the detection of Helicobacter pylori in stool specimens in the diagnosis of infection and posttreatment check-up. Am J Gastroenterol 95: 2200–2205. Islam S, et al. (2005) Stool antigen testing for the diagnosis and confirmation of eradication of Helicobacter pylori infection: a prospective blinded trial. Intern Med J 35:526–529. Kusters JG, et al. (1997) Coccoid forms of Helicobacter pylori are the morphologic manifestation of cell death. Infect Immun 65: 3672–3679. Makristathis A, et al. (2000) Two enzyme immunoassays and PCR for detection of Helicobacter pylori in stool specimens from pediatric patients before and after eradication therapy. J Clin Microbiol 38: 3710–3714. Malfertheiner P, et al. (2007) Current concepts in the management of Helicobacter pylori infection—the Maastricht III Consensus Report. Gut 56: 772–781. Ohkura R, et al. (2000) Usefulness of a novel enzyme immunoassay for the detection of Helicobacter pylori in feces. Scand J Gastroenterol 35: 49–53. Vaira D, et al. (1999) Diagnosis of Helicobacter pylori infection by HpSA test. European Helicobacter pylori HpSA Study Group. Lancet 354: 1732. Vaira D, et al. (2002a) Review article: Helicobacter pylori infection from pathogenesis to treatment—a critical reappraisal. Aliment Pharmacol Ther 16(Suppl 4):105–113.
S. Blanco et al. / Diagnostic Microbiology and Infectious Disease 63 (2009) 349–353 Vaira D, et al. (2002b) The stool antigen test for detection of Helicobacter pylori after eradication therapy. Ann Intern Med 136: 280–287. Veijola L, et al. (2005a) Stool antigen tests in the diagnosis of Helicobacter pylori infection before and after eradication therapy. World J Gastroenterol 11:7340–7344.
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Veijola L, et al. (2005b) Comparison of three stool antigen tests in confirming Helicobacter pylori eradication in adults. Scand J Gastroenterol 40: 395–401. Weingart V, et al. (2004) Sensitivity of a novel stool antigen test for detection of Helicobacter pylori in adult outpatients before and after eradication therapy. J Clin Microbiol 42:1319–1321.
Diagnostic Microbiology and Infectious Disease 63 (2009) 349 – 353 www.elsevier.com/locate/diagmicrobio
Bacteriology
Evaluation of a latex agglutination test (PYLOGEN) for the detection of Helicobacter pylori in stool specimens Silvia Blancoa,b , Montse Fornéc , Alicia Lacomaa,b , Cristina Prata,b , Miguel Ángel Cuestaa , Loreto Fuenzalidaa,b , Josep Maria Viverc , Jose Domíngueza,b,⁎ a
Servei de Microbiologia, Hospital Universitari Germans Trias i Pujol, Fundació Institut d'Investigació Germans Trias i Pujol, Badalona, 08916 Barcelona, Spain b Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain c Servei de Gastroenterologia, Hospital Universitari Mútua de Terrassa, 08221 Barcelona, Spain Received 17 October 2008; accepted 15 December 2008
Abstract The aim of the study was to assess a new latex agglutination (LA) stool antigen assay (PYLOGEN; CerTest Biotec, Zaragoza, Spain) in the diagnosis of Helicobacter pylori infection and to monitor its eradication after treatment. The LA test has been approved for sale in Europe, and its approval from the US Food and Drug Administration is still pending. The individuals enrolled were classified into 3 groups of patients: Group 1 consisted of 38 patients who are H. pylori positive. The diagnosis of H. pylori infection was established if there was concordance between 2 test results (urea breath test [UBT], rapid urease test, and histopathologic study) or if the culture alone was positive. Patients with only 1 positive test were considered indeterminate and were excluded from the study. Group 2 comprised 9 patients without positive tests and who were considered to be H. pylori negative. Group 3 consisted of 57 patients who received eradication treatment. The sensitivity and specificity of the test were 78.9% and 100%, respectively. The results of the UBT of the patients were studied 6 weeks after eradication therapy. The sensitivity and specificity of the LA test relative to UBT for patients after treatment were 75% and 93.3%, respectively. © 2009 Elsevier Inc. All rights reserved. Keywords: Helicobacter pylori; Stool antigen detection; Latex agglutination test; Monitoring eradication
1. Introduction Helicobacter pylori is recognized as one of the most common and medically important pathogens worldwide (Aksoy et al., 2003; Vaira et al., 2002a). In addition, it has been described as an etiologic agent in chronic gastritis and peptic ulcer disease and has also been associated with gastric malignancies (Asaka et al., 1994). Different invasive and noninvasive tests (Veijola et al., 2005b) are available for the diagnosis of H. pylori infection.
⁎ Corresponding author. Servei de Microbiologia, Fundació Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol, Ctra. del Canyet s/n, Badalona, 08916 Barcelona, Spain. Tel.: +34-934978894; fax: +34-934978895. E-mail address: [email protected] (J. Domínguez). 0732-8893/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2008.12.006
Although detection of H. pylori antigen in stool samples has been a subject of controversy, especially because of its poor correlation with the urea breath test (UBT) in the confirmation of eradication of H. pylori, it has been included in several clinical guidelines. Nowadays, stool antigen assays offer an alternative noninvasive method for the diagnosis of infection (Andrews et al., 2003; Forne et al., 2000; Islam et al., 2005; Vaira et al., 1999; Veijola et al., 2005a). Since the introduction of antigen stool assays, many studies have described the use of enzyme immunoassay (EIA) and immunochromatographic tests (ICTs) based on polyclonal or monoclonal antibodies for the diagnosis of H. pylori infections (Aksoy et al., 2003; Andrews et al., 2003; Blanco et al., 2008; Chisholm et al., 2004; Forne et al., 2000). Recently, the 1st latex agglutination (LA) assay for H. pylori antigen detection in stool samples has been developed. The aim of this study was to evaluate the
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usefulness of a new monoclonal antibody-based LA stool antigen assay (PYLOGEN; CerTest Biotec, Zaragoza, Spain) in the diagnosis of H. pylori infection and to monitor H. pylori eradication after treatment, comparing its accuracy with an ICT based on monoclonal antibody stool antigen assay (Letitest H. pylori CARD; Laboratorios Leti, Barcelona, Spain) and with 2 EIAs, one of them based on polyclonal antibodies (Premier Platinum HpSA; Meridian Diagnostics, Cincinnati, OH) and the other one based on monoclonal antibodies (Amplified IDEIA HpStAR; DakoCytomation, Cambridge, UK). 2. Methods 2.1. Patients The study population consisted of 104 patients reporting for routine upper gastrointestinal endoscopy at Hospital Universitari Mútua de Terrassa, Barcelona, Spain. Patients were selected retrospectively. Exclusion criteria were the recent use of bismuth-containing compounds or antibiotics (in the previous 2 months) or proton pump inhibitors (in the previous 15 days). The main diagnoses of the patients included ulcer (55.2%), antritis (18.4%), duodenitis (5.3%), and hiatal hernia (2.6%). UBT, rapid urease test (RUT), histopathologic study (H), culture in an antral biopsy specimen (Matrix test; Rohm Farm Laboratories, Germany), and detection of Helicobacter antigen by HpSA EIA test and HpStAR EIA were determined for all patients at the time initial diagnostic testing was performed. The diagnosis of H. pylori infection was established if there was concordance between 2 test results (RUT, UBT, or H) or if the culture alone was positive. Patients with only 1 positive test were considered indeterminate and were excluded from the study. We included patients during 2 stages of the treatment, at the time initial diagnostic testing was performed and 6 weeks after the patients finished the treatment, to evaluate the utility in assessing the eradication of the H. pylori. Individuals enrolled were classified into 3 groups: Group 1 consisted of 38 patients (14 female, mean age of 47.9 years) who are H. pylori positive (study group). Twelve patients had positive results for all tests (RUT, UBT, H, and culture), 18 patients had both results for RUT and H positive, and 8 patients had both results for RUT and UBT positive. Group 2 comprised 9 patients (3 female, mean age of 52.2 years) without any positive test (RUT, UBT, H, culture) who were considered to be H. pylori negative (control group). Group 3 consisted of 57 patients who received eradication treatment (2× omeprazole 20 mg, 2× clarithromycin 500 mg, and 2× amoxicillin 1 g for 7 days). The UBT results of the patients were studied after 6 weeks, and a stool sample was also collected to determine the presence of H. pylori antigen. In the posttreatment follow-up, UBT was used as the gold standard for considering eradication.
All samples were frozen at −70 °C before conducting the LA test and ICT; HpSA and HpStAR EIA tests were done before freezing and were not repeated. The study was approved by the ethics committees of the hospitals participating in the study. Patients expressed their consent to be included in the study. 2.2. Urea breath test UBT was performed in accordance with the European protocol (Dominguez-Munoz et al., 1997). Briefly, patients fasted overnight, and then a solution containing 4 g of citric acid was given before collecting baseline breath samples. Afterward, 100 mg of 13C-labeled urea dissolved in water was administered, and after 30 min, 2 breath samples were obtained. The increase in the mole fraction of tracer 13CO2 at 30 min compared with the baseline was expressed as atom percentage excess units. Of the results, more than 5% were considered positive, between 4.5% and 5% as indeterminate, and b4.5% as negative. 2.3. Stool antigen tests All the tests were performed, and the results were interpreted following the manufacturers' instructions. 2.4. PYLOGEN (CerTest Biotec) The latex reagent is a suspension of polystyrene latex particles of uniform size coated with monoclonal antibodies directed specifically against H. pylori antigen. The manufacture claims that the target antigen is of polysaccharide nature. Briefly, 100 μL of the latex reagent was mixed with 100 μL of diluted a stool sample on the slide. The results were read after a rotation of the slide for 10 min. The result was considered positive when the uniform appearance of the latex suspension converted to a clear agglutination. The LA test has been approved for sale in Europe, and its approval from the US Food and Drug Administration is still pending. 2.5. Letitest H. pylori CARD (Laboratorios Leti) The assay is an ICT based on monoclonal antibodies specific to H. pylori antigens. Five drops of a diluted sample were dispensed into the circular window marked with an arrow. The results were read after 10 min. If the result is negative, only 1 red band appears on the control line. If the sample is positive, a 2nd red band also appears in the sample line. 2.6. Premier Platinum HpSA EIA test (Meridian Diagnostics) The assay is based on polyclonal antibodies specific to H. pylori antigens. Diluted stool samples and a peroxidaseconjugated polyclonal antibody were added to the microwells. After washing the plate, substrate solution was added and incubated for 10 min at room temperature. The results were read by spectrophotometry. Absorbance (450/620 nm) ≥0.120 was considered positive.
S. Blanco et al. / Diagnostic Microbiology and Infectious Disease 63 (2009) 349–353
2.7. Amplified IDEIA™ HpStAR™ (DakoCytomation) The assay is an EIA based on monoclonal antibodies specific to H. pylori antigens. Diluted stool samples and a peroxidase-conjugated monoclonal antibody were added to the microwells and incubated for 1 h at room temperature. After washing the plate, the substrate solution was added and incubated for 10 min at room temperature. The results were read by spectrophotometric determination by means of an EIA microplate reader. An absorbance (450/620 nm) ≥0.150 was considered positive.
351
Table 2 Sensitivity and specificity of the stool antigen assays at the initial diagnosis of H. pylori infection and posttreatment follow-up Results antigen detection methods (%) LA Diagnosis Sensitivity 78.9 Specificity 100 Posttreatment follow-up Sensitivity 75 Specificity 93.3
Letitest ICT
HpSA EIA
HpStAR EIA
92.1 100
86.8 100
97.3 100
66.7 84.4
66.7 75.6
83.3 97.8
2.8. Statistical analysis Sensitivity and specificity were calculated in the usual manner. The agreement between different tests was estimated by Cohen's κ statistics. All analyses were made with SPSS statistical software for windows (SPSS version 15.0; SPSS, Chicago, IL). 3. Results Using LA, we detected H. pylori antigen in 30 of 38 samples from the study group (78.9% sensitivity) and in any of the patients from the control group (100% specificity). The sensitivity of Letitest ICT, HpStAR EIA, and HpSA EIA was 92.1%, 97.3%, and 86.8%, respectively. All the tests showed 100% specificity. In the 57 patients evaluated 6 weeks after the eradication therapy, the UBT result was negative in 45 patients, the LA test result was negative in 42 cases, the Letitest ICT result was negative in 38 cases, the HpSA EIA result was negative in 34, and the HpStAR EIA result was negative in 44 cases. The UBT result was positive for the remaining 12 patients, the LA test result was positive in 9 cases, the Letitest ICT result was positive in 8 cases, the HpSA EIA result was positive in 8 cases, and the HpStAR EIA result was positive in 10 cases. The overall agreements between the UBT and the antigen tests were as follows: LA, 89.4% (k = 0.683, SE =
Table 1 Results of the Helicobacter stool antigen methods in individuals infected and noninfected, at the time of diagnosis and after eradication therapy Results of the methods
LA Letitest ICT HpSA EIA HpStAR EIA
No. of individuals studied and their H. pylori status
Positive Negative Positive Negative Positive Negative Positive Negative
Before therapy
After therapy
Positive (n = 38)
Negative (n = 9)
Positive (n = 12)
Negative (n = 45)
30 8 35 3 33 5 37 1
0 9 0 9 0 9 0 9
9 3 8 4 8 4 10 2
3 42 7 38 9 34 1 44
0.120); Letitest ICT, 80.7% (k = 0.468; SE = 0.136); HpStAR EIA, 94.7% (k = 0.837; SE = 0.091); and HpSA EIA, 73.7% (k = 0.348; SE = 0.131). The sensitivity and specificity of LA test relative to UBT for patients after eradication therapy were 75% and 93.3%, respectively. The overall differences, including all group of patients, in the results between LA test and the Letitest ICT, HpSA EIA, and HpStAR EIA tests were not significant (P = 0.115, P = 0.089, P = 0.180, respectively). However, at the time initial diagnostic testing was performed, a significant difference only exists between the LA test and the HpStAR EIA test (P = 0.039). In the posttreatment follow-up, no significant differences between the antigen detection stool assays have been obtained. A summary of the results is shown in Tables 1 and 2.
4. Discussion The 1st antigen stool assay developed was the HpSA EIA assay, which uses polyclonal antibodies. This test has been extensively evaluated in pre- and posttreatment settings both in adults and children (Vaira et al., 2002b). It has been demonstrated that polyclonal assays are good tests for the diagnosis of infection but show low accuracy for monitoring eradication therapy (Aksoy et al., 2003; Forne et al., 2000; Islam et al., 2005; Ohkura et al., 2000; Vaira et al., 2002b). Previous evaluations comparing the polyclonal- and monoclonal-based assay results reveal comparable specificity but higher sensitivity of monoclonal tests for the diagnosis of H. pylori infection (Andrews et al., 2003; Asfeldt et al., 2004; Chisholm et al., 2004; Dominguez et al., 2006; Erzin et al., 2004; Weingart et al., 2004). As far as we know, this is the 1st time that an LA test has been evaluated for the diagnosis of H. pylori infection and eradication follow-up. This test has not been evaluated previously in comparison with other antigen tests. Our results show that the LA test is the less sensitive test among the tests evaluated for the initial diagnosis of H. pylori infection. On the contrary, among the antigen stool detection tests, the LA test and the HpStAR EIA are the more reliable methods, with the higher sensitivities and specificities, in monitoring the eradication after treatment. Nevertheless, in
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testing the eradication of the H. pylori at 6 weeks after finishing the treatment, any of the antigen stool detection tests evaluated had the same sensitivity as the UBT for determining persistent infection. The persistence of a positive antigen detection result in some patients with a negative UBT result 6 weeks after finishing the treatment has been explained by the longerlasting elimination of the low amounts of Helicobacter antigens from the degenerating remains of dead bacteria (Forne et al., 2000; Kusters et al., 1997). Therefore, the LA test, which had the lowest sensitivity at the diagnosis moment, has obtained better sensitivity and specificity than Letitest ICT and HpSA EIA in comparison with the UBT in patients who have eradicated the H. pylori. However, in our study, the test with a higher sensitivity (HpStAR) obtained the best agreement with the UBT. This fact could be explained by differences in the antigen detected. It has been hypothesized that HpStAR EIA may recognize a water-soluble antigen with a high turnover (Makristathis et al., 2000). The use of UBT alone may not be considered as the gold standard in the follow-up treatment. However, invasive diagnostic tests are not well tolerated by patients, and the value of UBT in assessing eradication of H. pylori has been clearly demonstrated. According to the Maastricht III Consensus Report (Malfertheiner et al., 2007), noninvasive tests should be used for confirmation of eradication, except in cases where repeat endoscopy is indicated, for example, patients with gastric ulcer. The main drawback of the current evaluation is that we studied a small group of patients. This fact could influence both the sensitivity and, specially, the specificity of the test evaluated. Another limitation is the lack of longitudinal follow-up in all the patients included at the moment of diagnosis. This was not possible because of logistical reasons. Despite these limitations, our study provides relevant data related to the clinical utility of the LA test for detection of H. pylori antigen in stool samples. Further studies including more patients could be adequate for confirming the results. LA assay is faster than the EIA, giving a result in a few minutes, and is easy to perform and interpret. LA test is comparable with ICT methods in technical complexity and the equipment required, allowing a wide diffusion of Helicobacter stool antigen detection in conventional laboratories, especially in areas with limited technical resources. Given that, some laboratories batch samples until they have enough to perform the EIA, causing delays in the diagnostic process. The LA test evaluated is especially useful for small hospital laboratories without appropriate equipment for performing the EIA and for laboratories that work with few samples. In conclusion, LA test is a method with a lower sensitivity than the other antigen assays tested, but with a high specificity to determine H. pylori infection. The test has 75% sensitivity and 93% specificity in comparison
with the UBT in monitoring Helicobacter eradication after therapy. Acknowledgments The authors would like to thank CerTest Biotec, Zaragoza, Spain, for providing the test kits required for the study. References Aksoy DY, et al. (2003) Evaluation of the Helicobacter pylori stool antigen test (HpSA) for the detection of Helicobacter pylori infection and comparison with other methods. Hepatogastroenterology 50: 1047–1049. Andrews J, et al. (2003) Comparison of three stool antigen tests for Helicobacter pylori detection. J Clin Pathol 56:769–771. Asaka M, et al. (1994) Possible role of Helicobacter pylori infection in early gastric cancer development. Cancer 73:2691–2694. Asfeldt A, et al. (2004) Accuracy of a monoclonal antibody-based stool antigen test in the diagnosis of Helicobacter pylori infection. Scand J Gastroenterol 39:1073–1077. Blanco S, et al. (2008) Comparison of stool antigen immunoassay methods for detecting Helicobacter pylori infection before and after eradication treatment. Diagn Microbiol Infect Dis 61:150–155. Chisholm SA, et al. (2004) Non-invasive diagnosis of Helicobacter pylori infection in adult dyspeptic patients by stool antigen detection: does the rapid immunochromatography test provide a reliable alternative to conventional ELISA kits? J Med Microbiol 53:623–627. Dominguez-Munoz JE, et al. (1997) A citric acid solution is an optimal test drink in the 13C-urea breath test for the diagnosis of Helicobacter pylori infection. Gut 40:459–462. Dominguez J, et al. (2006) Comparison of a monoclonal with a polyclonal antibody-based enzyme immunoassay stool test in diagnosing Helicobacter pylori infection before and after eradication therapy. Aliment Pharmacol Ther 23:1735–1740. Erzin Y, et al. (2004) Comparison of two different stool antigen tests for the primary diagnosis of Helicobacter pylori infection in Turkish patients with dyspepsia. Helicobacter 9:657–662. Forne M, et al. (2000) Accuracy of an enzyme immunoassay for the detection of Helicobacter pylori in stool specimens in the diagnosis of infection and posttreatment check-up. Am J Gastroenterol 95: 2200–2205. Islam S, et al. (2005) Stool antigen testing for the diagnosis and confirmation of eradication of Helicobacter pylori infection: a prospective blinded trial. Intern Med J 35:526–529. Kusters JG, et al. (1997) Coccoid forms of Helicobacter pylori are the morphologic manifestation of cell death. Infect Immun 65: 3672–3679. Makristathis A, et al. (2000) Two enzyme immunoassays and PCR for detection of Helicobacter pylori in stool specimens from pediatric patients before and after eradication therapy. J Clin Microbiol 38: 3710–3714. Malfertheiner P, et al. (2007) Current concepts in the management of Helicobacter pylori infection—the Maastricht III Consensus Report. Gut 56: 772–781. Ohkura R, et al. (2000) Usefulness of a novel enzyme immunoassay for the detection of Helicobacter pylori in feces. Scand J Gastroenterol 35: 49–53. Vaira D, et al. (1999) Diagnosis of Helicobacter pylori infection by HpSA test. European Helicobacter pylori HpSA Study Group. Lancet 354: 1732. Vaira D, et al. (2002a) Review article: Helicobacter pylori infection from pathogenesis to treatment—a critical reappraisal. Aliment Pharmacol Ther 16(Suppl 4):105–113.
S. Blanco et al. / Diagnostic Microbiology and Infectious Disease 63 (2009) 349–353 Vaira D, et al. (2002b) The stool antigen test for detection of Helicobacter pylori after eradication therapy. Ann Intern Med 136: 280–287. Veijola L, et al. (2005a) Stool antigen tests in the diagnosis of Helicobacter pylori infection before and after eradication therapy. World J Gastroenterol 11:7340–7344.
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Veijola L, et al. (2005b) Comparison of three stool antigen tests in confirming Helicobacter pylori eradication in adults. Scand J Gastroenterol 40: 395–401. Weingart V, et al. (2004) Sensitivity of a novel stool antigen test for detection of Helicobacter pylori in adult outpatients before and after eradication therapy. J Clin Microbiol 42:1319–1321.