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Evaluation of a new reagent strip rapid urease test for detection of Helicobacter pylori infection
Evaluation of a new reagent strip rapid urease test for detection of Helicobacter pylori infection Mahmoud M. Yousfi, MD, Hala M.T. El-Zimaity, MD, Robert M. Genta, MD, David Y. Graham, MD Houston, Texas
Background: Rapid urease tests are commonly used as a convenient method to detect Helicobacter pylori infection. Our previous experiments demonstrated enhanced efficacy of agar gel rapid urease test compared with reagent strip rapid urease tests. We evaluated the efficacy of PyloriTek, a new reagent strip rapid test for detecting H. pylori infection. Methods: Gastric antral mucosal biopsy specimens were obtained for comparison between agar gel rapid urease tests and PyloriTek (200 specimens). The rapid urease test to be used first was selected randomly. H. pylori status was determined using the Genta stain. Culture was performed to confirm H. pylori status when false rapid urease tests were suspected. Results: One hundred patients were studied; 68 had H. pylori infection. There were two false-negative and one false-positive PyloriTek when scored at 1 hour, compared with only one false-positive and no false-negative tests at 2 hours. With the agar gel rapid urease tests, there were no false-positive tests and 5 false-negative tests when scored at 1 hour, 2 false-negative tests at 12 hours and I at 24 hours; there were no false-positive tests. At I hour, 3% (95% CI = 1% to 9%) of PyloriTek tests had an erroneous categorization of H. pylori status compared with 5% for the agar gel rapid urease tests (95% CI = 1.6% to 11%) (p > 0.7). Conclusion: The new reagent strip rapid urease test, PyloriTek, is rapid and comparable in accuracy to agar gel rapid urease tests for detecting H. pylori infection. (Gastrointest Endosc 1996;44:519-22.) It is widely accepted t h a t antimicrobial therapy is indicated for all patients with Helicobacter pylori infection and peptic ulcer, i-3 The desire to detect the infection rapidly led to the development of several tests based on the high urease activity of H. pylori. 4-6 The rapid urease test (RUT) was introduced as a simple and convenient method t h a t often provides the endosReceived December 29, 1995. For revision March 18, 1996. Accepted April 29, 1996. From the Departments of Medicine and Pathology, the Veterans Affairs Medical Center, and the Division of Molecular Virology, Baylor College of Medicine, Houston, Texas. This work was supported by the Department of Veterans Affairs and by the generous support of Hilda Schwartz. PyloriTek and hpfast devices were kindly supplied without charge by their manufacturers. Reprint requests: David Y. Graham, MD, VAMC (111D), 2002 Holcombe Blvd., Houston, Texas 77030.
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copist with a diagnosis so that therapy need not be delayed until histology or bacterial culture results are available. 7-9 In the United States, there are three commercially available RUTs: CLOtest (Delta West Pty. Ltd., Australia) and hpfast (GI Supply, Camp Hill, Pa.) are agar gel type, and PyloriTek (Serim Research Corp., Elkhart, Ind.) is a reagent strip RUT. i°-i2 In a previous study, we compared PyloriTek and CLOtest for detection of H. pylori status, l° PyloriTek yielded an erroneous categorization of H. pylori status in 13.7% (95% CI = 7.7% to 22%) when the test was interpreted at 2 hours. The sensitivity was 98% but the specificity was only 68%. 1° When the test was interpreted at 1 hour the erroneous categorization rate was less (2.9%), but with two false-positive results, i° The PyloriTek test was modified to reduce the false-positive rate. We therefore evaluated the new PyloriTek test in a GASTROINTESTINAL ENDOSCOPY 5 1 9
Table 1. Comparison between PyloriTek and agar gel RUT for detection of H. pylori status
PyloriTek ~-hour reading 1-hour reading 2-hour reading Agar RUT ~-hour reading 1-hour reading 2-hour reading 4-hour reading 12-hour reading 24-hour reading
No.
TP
TN
FP
FN
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
FP + FN rate (%)
100 100 100
63 66 68
32 31 31
0 1 1
2 2 0
93 98.5 100
100 97 97
100 98.5 98.5
86.5 94 100
5 3 1
100 100 100 100 100 100
63 63 64 66 66 67
32 32 32 32 32 32
0 0 0 0 0 0
5 5 4 2 2 1
93 93 94 97 97 98.5
100 100 100 100 100 100
100 100 100 100 100 100
86 86 90 94 94 97
5 5 4 2 2 1
TP, True positive; FP, false positive; TN, true negative; FN, false negative; PPV, positive predictive value; NPV, negative predictive value.
prospective r a n d o m i z e d s t u d y in which we c o m p a r e d it w i t h a g a r rapid u r e a s e tests previously s h o w n to be essentially identical. 13
buffered formalin, cut in sequential 4 lam sections, stained with the Genta stain, 14 and examined by an experienced pathologist unaware of the patient's condition or endoscopy findings.
PATIENTS AND METHODS One hundred patients undergoing upper gastrointestinal endoscopy had gastric antral mucosal biopsies performed to determine their H. pylori status. The RUT used first was selected according to the patient's hospital number: PyloriTek first for odd numbers, and agar gel RUT first for even numbers. Biopsy specimens were taken prior to taking the specimens for histology. No patient had received a proton pump inhibitor, bismuth, or other antimicrobial within 4 weeks of evaluation. Antral mucosal biopsy specimens were taken from adjacent sites. The specimen was removed from the biopsy forceps with a needle. One of each was inserted into gel of the CLOtest or hpfast, then placed immediately in a warmer (Helicoview, GI Supply) at 36 ° C to 38 ° C and examined over the next 24 hours by one observer who was not blinded to the clinical data. Results were scored as positive at intervals less than ~ hour (0 to ~ hour), less than 1 hour (~ to 1 hour), less than 2 hours (1 to 2 hours), less than 4 hours (2 to 4 hours), less than 12 hours (4 to 12 hours), and at 24 hours. If no color change was observed after 24 hours for CLOtest or hpfast, the test was scored negative. Positive tests were recorded with a change in the CLOtest to pink and the hpfast to green. A second specimen was placed on a PyloriTek Reagent strip. After adding hydration reagent on the substrate pad Of the strip, the strip was folded and inserted into the reaction chamber and examined at three intervals: less than ~&hour (0 to ~4 hour); less than 1 hour (~ to 1 hour); and less than 2 hours (1 to 2 hours). The manufacturer recommends a 60minute observation period. The test was scored positive when observing a color change to blue over the specimen, similar to the color over the control. If no blue color appeared over the specimen, the PyloriTek test was scored negative. H. pylori status was documented by histopathologic examination. Eighty-eight patients had biopsy specimens taken from other sites in the antrum and from the gastric corpus for histopathology and/or culture, depending on their clinical scenario; 12 patients had only one antral biopsy specimen taken. Histology specimens were fixed with 10% 520 GASTROINTESTINAL ENDOSCOPY
RESULTS One h u n d r e d patients (72 m e n a n d 28 women) undergoing u p p e r g a s t r o i n t e s t i n a l endoscopy h a d a total of 200 gastric biopsies for rapid u r e a s e tests. Their ages r a n g e d from 29 to 81 y e a r s ( m e a n 52 years). Fifty h a d peptic ulcer disease (39 duodenal ulcer, 9 gastric ulcer, a n d 2 with both), 3 h a d mucosa-associated lymphoid tissue l y m p h o m a , 16 h a d dyspepsia without ulceration (usually g a s t r o e s o p h a g e a l reflux disease); 31 were n o r m a l volunteers. The prevalence of H. pylori infection was 68%. W h e n potential falsepositive or false-negative r a p i d u r e a s e tests were detected, the p e r t i n e n t biopsy slides were re-examined by a second pathologist to confirm their H. pylori status. There were no d i s a g r e e m e n t s b e t w e e n pathologists r e g a r d i n g the i n t e r p r e t a t i o n of these biopsies. I n all p a t i e n t s with false r a p i d u r e a s e tests, H. pylori s t a t u s was confirmed by serologic t e s t i n g for H. pylori (FlexSure H P test, S m i t h K l i n e Diagnostics, S a n Jose, Calif.) a n d by isolating the o r g a n i s m by culture.S, 15-17 The results were always consistent with histology. With a g a r gel RUTs (61 C L O t e s t s a n d 39 hpfasts), t h e r e was 1 false-negative (with CLOtest) a n d no false-positive tests. The erroneous categorization of H. pylori s t a t u s r a t e was 1% (95% C.I. = 0% to 5%) (Table 1). With PyloriTek t h e r e was one false-positive a n d two false-negative results at 1 hour; both false-negative results h a d converted to positive at 2 hours. The erroneous categorization of H. pylori s t a t u s u s i n g PyloriTek r a n g e d from 1% to 5% d e p e n d i n g on time of i n t e r p r e t a t i o n of the test results (Table 1). The sensitivity a n d specificity at 1 h o u r were 98% a n d 97%, respectively.
VOLUME 44, NO. 5, 1996
T h e r e w e r e no false-positive RUTs. At I h o u r t h e r e w e r e 5 f a l s e - n e g a t i v e tests, 4 of which c o n v e r t e d to t r u e - p o s i t i v e a t 24 h o u r s (Table 1). C o m p a r i s o n of results a t 1 h o u r s h o w e d t h a t t h e a g a r gel t e s t s accur a t e l y d e t e r m i n e d H. pylori s t a t u s in 92.6% of those w i t h H. pylori infection c o m p a r e d w i t h 95.5% w i t h t h e P y l o r i T e k (p = 0.71). At 2 h o u r s t h e correct categorization ofH. pylori s t a t u s w a s 96% a n d 99% for a g a r gel R U T c o m p a r e d w i t h PyloriTek, r e s p e c t i v e l y (p > 0.2), w i t h one false-positive P y l o r i T e k test.
DISCUSSION Although several invasive and noninvasive tests h a v e b e e n p r o v e n a c c u r a t e in detecting H. pylori infection, t h e histologic e x a m i n a t i o n of gastric biopsy s p e c i m e n s r e m a i n s the gold s t a n d a r d m e t h o d for diagnosis.S, 13, lS-25 H o w e v e r , histology is considered t i m e - c o n s u m i n g a n d in clinical practice a r a p i d diagnosis would be helpful to expedite t h e decision a b o u t i n i t i a t i n g anti-H, pylori t h e r a p y . 7, 26, 27 T h e r e are a n u m b e r of simple, accurate, a n d c o n v e n i e n t m e t h o d s for d i a g n o s i n g this infection, including serologic t e s t s for H. pylori, u r e a b r e a t h tests, a n d r a p i d u r e a s e tests. 27-33 Worldwide a n u m b e r of R U T s h a v e b e e n developed to help t h e endoscopists identify the p r e s e n c e of t h e b a c t e r i u m in t h e g a s t r i c mucosa. T h e R U T w a s introduced b y M c N u l t y et al. 34 I n o u r h a n d s , t h e C L O t e s t a n d t h e hpfast t e s t a g a r gel R U T s h a v e sensitivities a n d specificities e q u a l to or exceeding 90% w h e n u s e d e i t h e r before or a f t e r t h e r a p y for H. pylori infection.13, 35 We suspect t h a t lower diagnostic efficacy of R U T s in s o m e e a r l y r e p o r t s w a s due to d e c r e a s e d acc u r a c y of the m e t h o d s u s e d to diagnosis H. pylori infection. I n t r o d u c t i o n of n e w s t a i n s a n d c u l t u r e m e d i a h a v e i m p r o v e d the yield ofhistologic a n d microbiologic diagnosis so p a t h o l o g i s t s a n d microbiologists a r e able to b e t t e r identify t h e o r g a n i s m in t h e gastric specimens.14, 17 A g a r gel R U T s h a v e p r o v e n to be v e r y useful, r e s u l t i n g in e r r o n e o u s categorization o f H . pylori s t a t u s in only a b o u t 4% (95% CI = 1% to 9.7%) of cases w h e n scored a t 24 h o u r s a n d a p p r o x i m a t e l y 2% (95% C I = 0 . 2 % to 6.9%) w h e n scored a t less t h a n 12 hours. 13 T h e desire to i n c r e a s e t h e s p e e d of o b t a i n i n g a positive u r e a s e t e s t so as to provide t h e endoscopist w i t h t h e correct diagnosis before t h e p a t i e n t leaves t h e endoscopy suite led to t h e d e v e l o p m e n t of t h e r e a g e n t strip RUT. T h e original PyloriTek, while rapid, p r o v e d to be not v e r y reliable w h e n i n t e r p r e t e d a t 2 h o u r s as s u g g e s t e d b y t h e m a n u f a c t u r e r . T h e n e w version of t h e t e s t overcomes t h e p r o b l e m s w i t h the test, a n d int e r p r e t a t i o n a t 1 h o u r led to acceptable a c c u r a c y w i t h a n e r r o n e o u s c a t e g o r i z a t i o n o f H . pylori s t a t u s in only 2.9% (95% CI = 0.6% to 8.3%). P y l o r i T e k w a s developed to be i n t e r p r e t e d 1 h o u r a f t e r o b t a i n i n g t h e
VOLUME 44, NO. 5, 1996
biopsy s p e c i m e n a n d it h a s p r o v e d to be a r a p i d a n d reliable t e s t c o m p a r a b l e to t h e C L O t e s t a n d hpfast R U T s . U s e of a w a r m e r r e d u c e s t h e t i m e r e q u i r e d to detect a positive a g a r gel RUT, b u t the a d v a n t a g e g a i n e d in e a r l y detection is limited to t h e first 30 minu t e s of observation, s u g g e s t i n g t h a t t h e r e s u l t s would h a v e b e e n s i m i l a r w i t h o u t it. 36 We also u s e d j u m b o forceps. I t h a s p r e v i o u s l y b e e n s h o w n t h a t t h e s p e e d of t h e r e a c t i o n is i n c r e a s e d w h e n s e v e r a l biopsy speci m e n s (or one j u m b o biopsy) a r e placed in t h e a g a r gel. 37 I t r e m a i n s to be s e e n w h e t h e r t h e t i m e to interp r e t a b i l i t y of t h e a g a r gel R U T s a n d t h e r e a g e n t strip R U T would be s i m i l a r if single s p e c i m e n s o b t a i n e d w i t h e i t h e r s t a n d a r d or s m a l l - d i a m e t e r forceps a r e used.
REFERENCES 1. NIH Consensus Conference. Helicobacterpylori in peptic ulcer disease. NIH Consensus Development Panel on Helicobacter pylori in peptic ulcer disease. JAMA 1994;272:65-9. 2. Peura DA, Graham DY. Helicobacterpylori consensus reached: peptic ulcer is on the way to becoming an historic disease. Am J Gastroenterol 1994;89:1137-9. 3. Tytgat GNJ, Lee A, Graham DY, Dixon MF, Rokkas T. The role of infectious agents in peptic ulcer disease. Gastroenterol Int 1993;6:76-89. 4. Hazel] SL, Borody TJ, Gal A, Lee A. Campylobacterpyloridis gastritis I: detection ofurease as a marker of bacterial colonization and gastritis. Am J Gastroenterol 1987;82:292-6. 5. Szeto ML, Pounder RE, Hamilton-Dutoit SJ, Dhillon AP. Rapid urease test provides specific identification of Campylobacter pylori in antral mucosal biopsies. Postgrad Med J 1988;64: 935-6. 6. Marshall BJ, Warren JR, Francis GJ, Langton SR, Goodwin CS, Blincow ED. Rapid urease test in the management of Campylobacter pyloridis-associated gastritis. Am J Gastroenterol 1987;82:200-10. 7. Graham DY. Helicobacterpylori and the endoscopist: whether to diagnose. Gastrointest Endosc 1991;37:577-9. 8. Brown KE, Peura DA. Diagnosis ofHelicobacter pylori infection. Gastroenterol Clin North Am 1993;22:105-15. 9. Marshall BJ. Helicobacterpylori. Am J Gastroenterol 1994;89: Sl16-27. 10. Yousfi MM, E1-ZimaityHMT, Cole RC, Genta RM, Graham DY. Comparison of agar gel (CLOtest) or reagent strip (PyloriTek) by rapid urease tests for detection of Helicobacterpylori infection. Am J Gastroenterol 1995 (in press). 11. Ramirez F, Jackson FW. A new more specific rapid urease test to diagnose Helicobacter pylori gastritis. Gastrointest Endosc 1995;41:9-12. 12. Rogge JD, Wagner DR, Carrico RJ, etal. Evaluation of a new urease reagent strip for the detection of Helicobacterpylori in gastric biopsy specimens. Am J Gastroenterol 1995;90:1965-8. 13. Yousfi MM, E1-ZimaityHMT, Cole RC, Genta RM, Graham DY. Detection of Helicobacterpylori by rapid urease tests: is biopsy size a critical variable? Gastrointest Endosc 1996;43:222-4. 14. Genta RM, Robason GO, Graham DY. Simultaneous visualization of Helicobacterpylori and gastric morphology: a new stain. Hum Pathol 1994;25:221-6. 15. Young YL, Cutler AF. Evaluation of a rapid in officeserum test for Helicobacter pylori [abstract]. Gastroenterology 1995;108: A246. 16. Graham DY, Evans DG Jr, Peacock J, Baker JT, Schrier WH. Comparison of rapid serologic tests (Flexsure HP and QuickVue) with conventional ELISA for detection of Helicobacterpylori infection. Am J Gastroenterol 1996;91:942-8. 17. Hachem CY, Clarridge JE, Evans DG, Graham DY. Comparison of agar media for primary isolation of Helicobacterpylori. J Clin Pathol 1995;48:714-6. GASTROINTESTINAL ENDOSCOPY 521
18. Barthel JS, Everett ED. Diagnosis of Campylobacterpylori infection: the "gold standard" and the alternative. Rev Infect Dis 1990;12(suppl 1):$107-14. 19. Lin SK, Lambert JR, Schembri M, et al. A comparison of diagnostic tests to determine Helicobacter pylori infection. J Gastroenterol Hepatol 1992;7:203-9. 20. Schnell GA, Schubert TT. Usefulness of culture, histology, and urease testing in the detection of Campylobacter pylori. Am J Gastroenterol 1989;84:133-7. 21. Coudron PE, Kirby DF. Comparison of rapid urease tests, staining techniques, and growth on different solid media for detection of Campylobacter pylori. J Clin Microbiol 1989;27: 1527-30. 22. Deltenre M, Glupczynski Y, De Prez C, et al. The reliability of urease tests, histology and culture in the diagnosis of Campylobacter pylori infection. Scand J Gastreenterol Suppl 1989; 160:19-24. 23. Chodos JE, Dworkin BM, Smith F, Van Horn K, Weiss L, Rosenthal WS. Campylobacter pylori and gastroduodenal disease: a prospective endoscopic study and comparison of diagnostic tests. Am J Gastroenterol 1988;83:1226-30. 24. Alpert LC, Graham DY, Evans DJ Jr, et al. Diagnostic possibilities for Campylobacter pylori infection. Eur J Gastroenterol Hepatol 1989;1:17-26. 25. Conti-Nibali S, Sferlazzas C, Fera MT, Saitta G, Tedeschi A, Magazzu G. Helicobacterpylori infection: a simplified diagnostic approach. Am J Gastroenterol 1990;85:1573-5. 26. Graham DY, BSrsch GM. The who's and when's of therapy for HelicobaCter pylori. Am J Gastroenterol 1990;85:1552-5. 27. Peura DA. Helicobacterpylori: a diagnostic dilemma and a dilemma of diagnosis. Gastroenterology 1995;109:313-5. 28. Malfertheiner P. Diagnosis of Helicobacterpylori infection. In: Axon ART, editor. Helicobacterpylori: its role in gastrointestinal disease. London: Science Press, 1994:11-7.
29. Westblom TU, Madan E, Kemp J, Subik MA. Evaluation of a rapid urease test to detect Campylobacter pylori infection. J Clin Microbiol 1988;26:1393-4. 30. Abdalla S, Marco F, Perez RM, et al. Rapid detection of gastric Campylobacterpylori colonization by a simple biochemical test. J Clin Microbiol 1989;27:2604-5. 31. Kolts BE, Joseph B, Achem SR, Bianchi T, Monteiro C. Helicobacterpylori detection: a quality and cost analysis. Am J Gastroenterol 1993;88:650-5. 32. Katelaris PH, Lowe DG, Norbu P, Farthing MJ. Field evaluation of a rapid, simple and inexpensive urease test for the detection ofHelicobacterpylori. J Gastroenterol Hepatol 1992;7: 569-71. 33. Cutler AF, Havstad S, Ma CK, Blaser MJ, Perez-Perez GI, Schubert TT. Accuracy of invasive and noninvasivetests to diagnose Helicobacter pylori infection. Gastroenterology 1995; 109:136-41. 34. McNulty CA, Dent JC, UffJS, Gear MW, Wilkinson SP. Detection of Campylobacter pylori by the biopsy urease test: an assessment in 1445 patients. Gut 1989;30:1058-62. 35. E1-Zimaity HMT, A1-AssiMT, Genta RM, Graham DY. Confirmation of successful therapy of Helicobacter pylori infection: number and site of biopsies or a rapid urease test. Am J Gastroenterol 1995;90:1962-5. 36. Yousfi MM, E1-Zimaity HMT, Cole RC, Genta RM, Graham DY. Does using a warmer influence the results of rapid urease testing for Helicobacter pylori? Gastrointest Endosc 1996;43: 260-1. 37. Laine L, Chun D, Stein C, E1-Beblawi I, Sharma V, Chandrasoma P. The influence of size and number of biopsies on rapid urease test results: a prospective evaluation. Gastrointest Endosc 1996;43:49-53.
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522 GASTROINTESTINAL ENDOSCOPY
VOLUME 44, NO. 5, 1996
Background: Rapid urease tests are commonly used as a convenient method to detect Helicobacter pylori infection. Our previous experiments demonstrated enhanced efficacy of agar gel rapid urease test compared with reagent strip rapid urease tests. We evaluated the efficacy of PyloriTek, a new reagent strip rapid test for detecting H. pylori infection. Methods: Gastric antral mucosal biopsy specimens were obtained for comparison between agar gel rapid urease tests and PyloriTek (200 specimens). The rapid urease test to be used first was selected randomly. H. pylori status was determined using the Genta stain. Culture was performed to confirm H. pylori status when false rapid urease tests were suspected. Results: One hundred patients were studied; 68 had H. pylori infection. There were two false-negative and one false-positive PyloriTek when scored at 1 hour, compared with only one false-positive and no false-negative tests at 2 hours. With the agar gel rapid urease tests, there were no false-positive tests and 5 false-negative tests when scored at 1 hour, 2 false-negative tests at 12 hours and I at 24 hours; there were no false-positive tests. At I hour, 3% (95% CI = 1% to 9%) of PyloriTek tests had an erroneous categorization of H. pylori status compared with 5% for the agar gel rapid urease tests (95% CI = 1.6% to 11%) (p > 0.7). Conclusion: The new reagent strip rapid urease test, PyloriTek, is rapid and comparable in accuracy to agar gel rapid urease tests for detecting H. pylori infection. (Gastrointest Endosc 1996;44:519-22.) It is widely accepted t h a t antimicrobial therapy is indicated for all patients with Helicobacter pylori infection and peptic ulcer, i-3 The desire to detect the infection rapidly led to the development of several tests based on the high urease activity of H. pylori. 4-6 The rapid urease test (RUT) was introduced as a simple and convenient method t h a t often provides the endosReceived December 29, 1995. For revision March 18, 1996. Accepted April 29, 1996. From the Departments of Medicine and Pathology, the Veterans Affairs Medical Center, and the Division of Molecular Virology, Baylor College of Medicine, Houston, Texas. This work was supported by the Department of Veterans Affairs and by the generous support of Hilda Schwartz. PyloriTek and hpfast devices were kindly supplied without charge by their manufacturers. Reprint requests: David Y. Graham, MD, VAMC (111D), 2002 Holcombe Blvd., Houston, Texas 77030.
37/1/74542 VOLUME 44, NO. 5, 1996
copist with a diagnosis so that therapy need not be delayed until histology or bacterial culture results are available. 7-9 In the United States, there are three commercially available RUTs: CLOtest (Delta West Pty. Ltd., Australia) and hpfast (GI Supply, Camp Hill, Pa.) are agar gel type, and PyloriTek (Serim Research Corp., Elkhart, Ind.) is a reagent strip RUT. i°-i2 In a previous study, we compared PyloriTek and CLOtest for detection of H. pylori status, l° PyloriTek yielded an erroneous categorization of H. pylori status in 13.7% (95% CI = 7.7% to 22%) when the test was interpreted at 2 hours. The sensitivity was 98% but the specificity was only 68%. 1° When the test was interpreted at 1 hour the erroneous categorization rate was less (2.9%), but with two false-positive results, i° The PyloriTek test was modified to reduce the false-positive rate. We therefore evaluated the new PyloriTek test in a GASTROINTESTINAL ENDOSCOPY 5 1 9
Table 1. Comparison between PyloriTek and agar gel RUT for detection of H. pylori status
PyloriTek ~-hour reading 1-hour reading 2-hour reading Agar RUT ~-hour reading 1-hour reading 2-hour reading 4-hour reading 12-hour reading 24-hour reading
No.
TP
TN
FP
FN
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
FP + FN rate (%)
100 100 100
63 66 68
32 31 31
0 1 1
2 2 0
93 98.5 100
100 97 97
100 98.5 98.5
86.5 94 100
5 3 1
100 100 100 100 100 100
63 63 64 66 66 67
32 32 32 32 32 32
0 0 0 0 0 0
5 5 4 2 2 1
93 93 94 97 97 98.5
100 100 100 100 100 100
100 100 100 100 100 100
86 86 90 94 94 97
5 5 4 2 2 1
TP, True positive; FP, false positive; TN, true negative; FN, false negative; PPV, positive predictive value; NPV, negative predictive value.
prospective r a n d o m i z e d s t u d y in which we c o m p a r e d it w i t h a g a r rapid u r e a s e tests previously s h o w n to be essentially identical. 13
buffered formalin, cut in sequential 4 lam sections, stained with the Genta stain, 14 and examined by an experienced pathologist unaware of the patient's condition or endoscopy findings.
PATIENTS AND METHODS One hundred patients undergoing upper gastrointestinal endoscopy had gastric antral mucosal biopsies performed to determine their H. pylori status. The RUT used first was selected according to the patient's hospital number: PyloriTek first for odd numbers, and agar gel RUT first for even numbers. Biopsy specimens were taken prior to taking the specimens for histology. No patient had received a proton pump inhibitor, bismuth, or other antimicrobial within 4 weeks of evaluation. Antral mucosal biopsy specimens were taken from adjacent sites. The specimen was removed from the biopsy forceps with a needle. One of each was inserted into gel of the CLOtest or hpfast, then placed immediately in a warmer (Helicoview, GI Supply) at 36 ° C to 38 ° C and examined over the next 24 hours by one observer who was not blinded to the clinical data. Results were scored as positive at intervals less than ~ hour (0 to ~ hour), less than 1 hour (~ to 1 hour), less than 2 hours (1 to 2 hours), less than 4 hours (2 to 4 hours), less than 12 hours (4 to 12 hours), and at 24 hours. If no color change was observed after 24 hours for CLOtest or hpfast, the test was scored negative. Positive tests were recorded with a change in the CLOtest to pink and the hpfast to green. A second specimen was placed on a PyloriTek Reagent strip. After adding hydration reagent on the substrate pad Of the strip, the strip was folded and inserted into the reaction chamber and examined at three intervals: less than ~&hour (0 to ~4 hour); less than 1 hour (~ to 1 hour); and less than 2 hours (1 to 2 hours). The manufacturer recommends a 60minute observation period. The test was scored positive when observing a color change to blue over the specimen, similar to the color over the control. If no blue color appeared over the specimen, the PyloriTek test was scored negative. H. pylori status was documented by histopathologic examination. Eighty-eight patients had biopsy specimens taken from other sites in the antrum and from the gastric corpus for histopathology and/or culture, depending on their clinical scenario; 12 patients had only one antral biopsy specimen taken. Histology specimens were fixed with 10% 520 GASTROINTESTINAL ENDOSCOPY
RESULTS One h u n d r e d patients (72 m e n a n d 28 women) undergoing u p p e r g a s t r o i n t e s t i n a l endoscopy h a d a total of 200 gastric biopsies for rapid u r e a s e tests. Their ages r a n g e d from 29 to 81 y e a r s ( m e a n 52 years). Fifty h a d peptic ulcer disease (39 duodenal ulcer, 9 gastric ulcer, a n d 2 with both), 3 h a d mucosa-associated lymphoid tissue l y m p h o m a , 16 h a d dyspepsia without ulceration (usually g a s t r o e s o p h a g e a l reflux disease); 31 were n o r m a l volunteers. The prevalence of H. pylori infection was 68%. W h e n potential falsepositive or false-negative r a p i d u r e a s e tests were detected, the p e r t i n e n t biopsy slides were re-examined by a second pathologist to confirm their H. pylori status. There were no d i s a g r e e m e n t s b e t w e e n pathologists r e g a r d i n g the i n t e r p r e t a t i o n of these biopsies. I n all p a t i e n t s with false r a p i d u r e a s e tests, H. pylori s t a t u s was confirmed by serologic t e s t i n g for H. pylori (FlexSure H P test, S m i t h K l i n e Diagnostics, S a n Jose, Calif.) a n d by isolating the o r g a n i s m by culture.S, 15-17 The results were always consistent with histology. With a g a r gel RUTs (61 C L O t e s t s a n d 39 hpfasts), t h e r e was 1 false-negative (with CLOtest) a n d no false-positive tests. The erroneous categorization of H. pylori s t a t u s r a t e was 1% (95% C.I. = 0% to 5%) (Table 1). With PyloriTek t h e r e was one false-positive a n d two false-negative results at 1 hour; both false-negative results h a d converted to positive at 2 hours. The erroneous categorization of H. pylori s t a t u s u s i n g PyloriTek r a n g e d from 1% to 5% d e p e n d i n g on time of i n t e r p r e t a t i o n of the test results (Table 1). The sensitivity a n d specificity at 1 h o u r were 98% a n d 97%, respectively.
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T h e r e w e r e no false-positive RUTs. At I h o u r t h e r e w e r e 5 f a l s e - n e g a t i v e tests, 4 of which c o n v e r t e d to t r u e - p o s i t i v e a t 24 h o u r s (Table 1). C o m p a r i s o n of results a t 1 h o u r s h o w e d t h a t t h e a g a r gel t e s t s accur a t e l y d e t e r m i n e d H. pylori s t a t u s in 92.6% of those w i t h H. pylori infection c o m p a r e d w i t h 95.5% w i t h t h e P y l o r i T e k (p = 0.71). At 2 h o u r s t h e correct categorization ofH. pylori s t a t u s w a s 96% a n d 99% for a g a r gel R U T c o m p a r e d w i t h PyloriTek, r e s p e c t i v e l y (p > 0.2), w i t h one false-positive P y l o r i T e k test.
DISCUSSION Although several invasive and noninvasive tests h a v e b e e n p r o v e n a c c u r a t e in detecting H. pylori infection, t h e histologic e x a m i n a t i o n of gastric biopsy s p e c i m e n s r e m a i n s the gold s t a n d a r d m e t h o d for diagnosis.S, 13, lS-25 H o w e v e r , histology is considered t i m e - c o n s u m i n g a n d in clinical practice a r a p i d diagnosis would be helpful to expedite t h e decision a b o u t i n i t i a t i n g anti-H, pylori t h e r a p y . 7, 26, 27 T h e r e are a n u m b e r of simple, accurate, a n d c o n v e n i e n t m e t h o d s for d i a g n o s i n g this infection, including serologic t e s t s for H. pylori, u r e a b r e a t h tests, a n d r a p i d u r e a s e tests. 27-33 Worldwide a n u m b e r of R U T s h a v e b e e n developed to help t h e endoscopists identify the p r e s e n c e of t h e b a c t e r i u m in t h e g a s t r i c mucosa. T h e R U T w a s introduced b y M c N u l t y et al. 34 I n o u r h a n d s , t h e C L O t e s t a n d t h e hpfast t e s t a g a r gel R U T s h a v e sensitivities a n d specificities e q u a l to or exceeding 90% w h e n u s e d e i t h e r before or a f t e r t h e r a p y for H. pylori infection.13, 35 We suspect t h a t lower diagnostic efficacy of R U T s in s o m e e a r l y r e p o r t s w a s due to d e c r e a s e d acc u r a c y of the m e t h o d s u s e d to diagnosis H. pylori infection. I n t r o d u c t i o n of n e w s t a i n s a n d c u l t u r e m e d i a h a v e i m p r o v e d the yield ofhistologic a n d microbiologic diagnosis so p a t h o l o g i s t s a n d microbiologists a r e able to b e t t e r identify t h e o r g a n i s m in t h e gastric specimens.14, 17 A g a r gel R U T s h a v e p r o v e n to be v e r y useful, r e s u l t i n g in e r r o n e o u s categorization o f H . pylori s t a t u s in only a b o u t 4% (95% CI = 1% to 9.7%) of cases w h e n scored a t 24 h o u r s a n d a p p r o x i m a t e l y 2% (95% C I = 0 . 2 % to 6.9%) w h e n scored a t less t h a n 12 hours. 13 T h e desire to i n c r e a s e t h e s p e e d of o b t a i n i n g a positive u r e a s e t e s t so as to provide t h e endoscopist w i t h t h e correct diagnosis before t h e p a t i e n t leaves t h e endoscopy suite led to t h e d e v e l o p m e n t of t h e r e a g e n t strip RUT. T h e original PyloriTek, while rapid, p r o v e d to be not v e r y reliable w h e n i n t e r p r e t e d a t 2 h o u r s as s u g g e s t e d b y t h e m a n u f a c t u r e r . T h e n e w version of t h e t e s t overcomes t h e p r o b l e m s w i t h the test, a n d int e r p r e t a t i o n a t 1 h o u r led to acceptable a c c u r a c y w i t h a n e r r o n e o u s c a t e g o r i z a t i o n o f H . pylori s t a t u s in only 2.9% (95% CI = 0.6% to 8.3%). P y l o r i T e k w a s developed to be i n t e r p r e t e d 1 h o u r a f t e r o b t a i n i n g t h e
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biopsy s p e c i m e n a n d it h a s p r o v e d to be a r a p i d a n d reliable t e s t c o m p a r a b l e to t h e C L O t e s t a n d hpfast R U T s . U s e of a w a r m e r r e d u c e s t h e t i m e r e q u i r e d to detect a positive a g a r gel RUT, b u t the a d v a n t a g e g a i n e d in e a r l y detection is limited to t h e first 30 minu t e s of observation, s u g g e s t i n g t h a t t h e r e s u l t s would h a v e b e e n s i m i l a r w i t h o u t it. 36 We also u s e d j u m b o forceps. I t h a s p r e v i o u s l y b e e n s h o w n t h a t t h e s p e e d of t h e r e a c t i o n is i n c r e a s e d w h e n s e v e r a l biopsy speci m e n s (or one j u m b o biopsy) a r e placed in t h e a g a r gel. 37 I t r e m a i n s to be s e e n w h e t h e r t h e t i m e to interp r e t a b i l i t y of t h e a g a r gel R U T s a n d t h e r e a g e n t strip R U T would be s i m i l a r if single s p e c i m e n s o b t a i n e d w i t h e i t h e r s t a n d a r d or s m a l l - d i a m e t e r forceps a r e used.
REFERENCES 1. NIH Consensus Conference. Helicobacterpylori in peptic ulcer disease. NIH Consensus Development Panel on Helicobacter pylori in peptic ulcer disease. JAMA 1994;272:65-9. 2. Peura DA, Graham DY. Helicobacterpylori consensus reached: peptic ulcer is on the way to becoming an historic disease. Am J Gastroenterol 1994;89:1137-9. 3. Tytgat GNJ, Lee A, Graham DY, Dixon MF, Rokkas T. The role of infectious agents in peptic ulcer disease. Gastroenterol Int 1993;6:76-89. 4. Hazel] SL, Borody TJ, Gal A, Lee A. Campylobacterpyloridis gastritis I: detection ofurease as a marker of bacterial colonization and gastritis. Am J Gastroenterol 1987;82:292-6. 5. Szeto ML, Pounder RE, Hamilton-Dutoit SJ, Dhillon AP. Rapid urease test provides specific identification of Campylobacter pylori in antral mucosal biopsies. Postgrad Med J 1988;64: 935-6. 6. Marshall BJ, Warren JR, Francis GJ, Langton SR, Goodwin CS, Blincow ED. Rapid urease test in the management of Campylobacter pyloridis-associated gastritis. Am J Gastroenterol 1987;82:200-10. 7. Graham DY. Helicobacterpylori and the endoscopist: whether to diagnose. Gastrointest Endosc 1991;37:577-9. 8. Brown KE, Peura DA. Diagnosis ofHelicobacter pylori infection. Gastroenterol Clin North Am 1993;22:105-15. 9. Marshall BJ. Helicobacterpylori. Am J Gastroenterol 1994;89: Sl16-27. 10. Yousfi MM, E1-ZimaityHMT, Cole RC, Genta RM, Graham DY. Comparison of agar gel (CLOtest) or reagent strip (PyloriTek) by rapid urease tests for detection of Helicobacterpylori infection. Am J Gastroenterol 1995 (in press). 11. Ramirez F, Jackson FW. A new more specific rapid urease test to diagnose Helicobacter pylori gastritis. Gastrointest Endosc 1995;41:9-12. 12. Rogge JD, Wagner DR, Carrico RJ, etal. Evaluation of a new urease reagent strip for the detection of Helicobacterpylori in gastric biopsy specimens. Am J Gastroenterol 1995;90:1965-8. 13. Yousfi MM, E1-ZimaityHMT, Cole RC, Genta RM, Graham DY. Detection of Helicobacterpylori by rapid urease tests: is biopsy size a critical variable? Gastrointest Endosc 1996;43:222-4. 14. Genta RM, Robason GO, Graham DY. Simultaneous visualization of Helicobacterpylori and gastric morphology: a new stain. Hum Pathol 1994;25:221-6. 15. Young YL, Cutler AF. Evaluation of a rapid in officeserum test for Helicobacter pylori [abstract]. Gastroenterology 1995;108: A246. 16. Graham DY, Evans DG Jr, Peacock J, Baker JT, Schrier WH. Comparison of rapid serologic tests (Flexsure HP and QuickVue) with conventional ELISA for detection of Helicobacterpylori infection. Am J Gastroenterol 1996;91:942-8. 17. Hachem CY, Clarridge JE, Evans DG, Graham DY. Comparison of agar media for primary isolation of Helicobacterpylori. J Clin Pathol 1995;48:714-6. GASTROINTESTINAL ENDOSCOPY 521
18. Barthel JS, Everett ED. Diagnosis of Campylobacterpylori infection: the "gold standard" and the alternative. Rev Infect Dis 1990;12(suppl 1):$107-14. 19. Lin SK, Lambert JR, Schembri M, et al. A comparison of diagnostic tests to determine Helicobacter pylori infection. J Gastroenterol Hepatol 1992;7:203-9. 20. Schnell GA, Schubert TT. Usefulness of culture, histology, and urease testing in the detection of Campylobacter pylori. Am J Gastroenterol 1989;84:133-7. 21. Coudron PE, Kirby DF. Comparison of rapid urease tests, staining techniques, and growth on different solid media for detection of Campylobacter pylori. J Clin Microbiol 1989;27: 1527-30. 22. Deltenre M, Glupczynski Y, De Prez C, et al. The reliability of urease tests, histology and culture in the diagnosis of Campylobacter pylori infection. Scand J Gastreenterol Suppl 1989; 160:19-24. 23. Chodos JE, Dworkin BM, Smith F, Van Horn K, Weiss L, Rosenthal WS. Campylobacter pylori and gastroduodenal disease: a prospective endoscopic study and comparison of diagnostic tests. Am J Gastroenterol 1988;83:1226-30. 24. Alpert LC, Graham DY, Evans DJ Jr, et al. Diagnostic possibilities for Campylobacter pylori infection. Eur J Gastroenterol Hepatol 1989;1:17-26. 25. Conti-Nibali S, Sferlazzas C, Fera MT, Saitta G, Tedeschi A, Magazzu G. Helicobacterpylori infection: a simplified diagnostic approach. Am J Gastroenterol 1990;85:1573-5. 26. Graham DY, BSrsch GM. The who's and when's of therapy for HelicobaCter pylori. Am J Gastroenterol 1990;85:1552-5. 27. Peura DA. Helicobacterpylori: a diagnostic dilemma and a dilemma of diagnosis. Gastroenterology 1995;109:313-5. 28. Malfertheiner P. Diagnosis of Helicobacterpylori infection. In: Axon ART, editor. Helicobacterpylori: its role in gastrointestinal disease. London: Science Press, 1994:11-7.
29. Westblom TU, Madan E, Kemp J, Subik MA. Evaluation of a rapid urease test to detect Campylobacter pylori infection. J Clin Microbiol 1988;26:1393-4. 30. Abdalla S, Marco F, Perez RM, et al. Rapid detection of gastric Campylobacterpylori colonization by a simple biochemical test. J Clin Microbiol 1989;27:2604-5. 31. Kolts BE, Joseph B, Achem SR, Bianchi T, Monteiro C. Helicobacterpylori detection: a quality and cost analysis. Am J Gastroenterol 1993;88:650-5. 32. Katelaris PH, Lowe DG, Norbu P, Farthing MJ. Field evaluation of a rapid, simple and inexpensive urease test for the detection ofHelicobacterpylori. J Gastroenterol Hepatol 1992;7: 569-71. 33. Cutler AF, Havstad S, Ma CK, Blaser MJ, Perez-Perez GI, Schubert TT. Accuracy of invasive and noninvasivetests to diagnose Helicobacter pylori infection. Gastroenterology 1995; 109:136-41. 34. McNulty CA, Dent JC, UffJS, Gear MW, Wilkinson SP. Detection of Campylobacter pylori by the biopsy urease test: an assessment in 1445 patients. Gut 1989;30:1058-62. 35. E1-Zimaity HMT, A1-AssiMT, Genta RM, Graham DY. Confirmation of successful therapy of Helicobacter pylori infection: number and site of biopsies or a rapid urease test. Am J Gastroenterol 1995;90:1962-5. 36. Yousfi MM, E1-Zimaity HMT, Cole RC, Genta RM, Graham DY. Does using a warmer influence the results of rapid urease testing for Helicobacter pylori? Gastrointest Endosc 1996;43: 260-1. 37. Laine L, Chun D, Stein C, E1-Beblawi I, Sharma V, Chandrasoma P. The influence of size and number of biopsies on rapid urease test results: a prospective evaluation. Gastrointest Endosc 1996;43:49-53.
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