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Serologic diagnosis of Helicobacter pylori-associated gastrointestinal disease
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phia, JB Lippincott, 1988, pp. 692-700. 34. Rump JA, Woemer I, Roth M, et al.: A new type of ANCA in active ulcerative colitis: Correlation to therapy and disease activity. In: Gross WL (ed): Advances in Experimental Medicine and Biology. Abstract Book of the 4th International Workshop on ANCA. London, Plenum, 1992, p. 36. 35. Sachar DB: Ulcerative colitis and sclerosing cholangitis: Does "IBD" mean "Inflamed Bile Ducts"? (editorial). Gastroenterology 100:14691470, 1991. 36. Saxon A, Shanahan F, Landers C, et al.: A distinct subset of antineutrophil cytoplasmic antibodies is associated with inflammatory bowel
37.
38.
39.
40.
disease. J Allergy Clin Immunol 86:202-210, 1990. Seibold F, Weber P, Klein R, et al.: Clinical significance of antibodies against neutrophils in patients with inflanunatory bowel disease and primary sclerosing cholangitis. Gut 33:657-662, 1992. Shanahan F, Duerr R, Rotter Yl, et al.: Neutrophil autoantibodies in ulcerative colitis: Familial aggregation and genetic heterogeneity. Gastroenterology 103:456-461, 1992. Shanahan F, Landers C, Duerr R: Neutrophil autoantibodies as disease markers for ulcerative colitis. Immunol Res 10:479-484, 1991. Snook JA, Chapman RW, Fleming K, et al.: Anti-
neutrophil nuclear antibody in ulcerative colitis, Crohn's disease and primary sclerosing cholangitis. Clin Exp Inununol 76:30--33, 1989. 41. Wiik A: Delineation of a standard method for indirect immunofluorescence detection of ANCA. Acta Pathol Microbiol Scand 97(Suppl 6):12-13, 1989. 42, Zauli D, Baffoni L, Cassani F, et al.: Antineutrophil cytoplasmic antibodies in primary sclerosing cholangitis, ulcerative colitis, and autoinunune diseases (letter). Gastroenterology 102:10881089, 1992.
Serologic Diagnosis of H e l i c o b a c t e r pylori-Associated Gastrointestinal Disease Richard A. Venezia and Jeffrey S. Ross Department of Pathology and Laboratory Medicine, Albany Medical Center, Albany, New York
H
"elicobacter pylori causes a slow,
human gastroduodenal infection ,with a high infection rate that is asymptomatic in many patients. Gastric bacteria were first described in 1874, and over the next century endogenous human gastric urease activity was found to be associated with spiral-shaped intragastric organisms. A spiral bacterium was isolated and cultured in 1983 from the human stomach by Warren and Marshall. 39 Since then, a considerable volume of literature as to the nature of the organism and its potential role in human disease has been published. H. pylori has been isolated throughout the world. The normal prevalence rate varies from 0% to nearly 50% in any geographic site and socioeconomic status. 34In the United States, H. pylori prevalence during the first four decades of life increases from 0% to 10% and reaches 50% beyond the fifth and sixth decades. In Third World countries, a 10% prevalence in the first
decade of life many reach 50% in the second decade and 75% by the fourth decade. Microbiologic Features H. pylori is a Gram-negative coccobacillary to spiral-shaped bacterium with four to six unipolar sheathed flagellae. It is fastidious, requiring a warm microaerobic environment, and has a strong affinity for human gastric-type epithelium. 15H. pylori may be isolated from the human stomach, containing normal or increased acid content, relatively easily using a variety of liquid and solid media featuring antibiotic supplements to control growth of competing organismsY 4,29,3~In the achlorhydric specimen, special techniques may be necessary as organisms other than H. pylori may colonize the human stomach in these conditions. The most characteristic biochemical property of H. pylori is urease production, which distinguishes it from the Campylobacter species and other related bacteria, z5
© 1993 Elsevier Science Publishing Co., Inc.
Although the 35-38 M percent guanosineplus-cytosine value for H. pylori places the organism in the range of the Campylobacter species, H. pylori is separated from Campylobacter by urease production, ultrastructural features, and fatty acid composition. The rRNA sequences of H. pylori are also different from Campylobacter as defined from the original Australian isolates and therefore warrants placement of this bacterium in the Helicobacter genus.
Human Gastrointestinal Disease Associated with H. pylori H. pylori has been isolated from normal human gastric antrum, duodenal metaplasia, and Meckel's diverticulum, but rarely from the rectum.4,7,32The organism has been isolated from 83% of patients with chronic superficial gastritis, 92% of patients with duodenal ulcer, 69% of patients with gastric ulcers, and 51% of patients with nonulcer dyspepsia. The organism has not been isolated generally
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126 CLINICAL I M M U N O L O G Y Newsletter
from esophageal disorders, nor is it identified in most cases of alrophic gastritis or nonsteroidal antiinflammatory drug-related gastritis. Relapse rates in duodenal ulcers have shown that H. pylori positive patients suffer relapse in 75%--95% of cases as compared with H. pylori negative patients with a less than 25% duodenal ulcer relapse rate. H. pylori has been linked in several major epidemiologic studies to the risk of gastric adenocarcinoma and lymphoma. TM The odds ratio of gastric cancer has ranged from 2.7 to 6.0. However, experimental animal and prospective clinical studies have not, as yet, defined a cause-effect relationship between the organism and precancerous and cancerous changes in the gastric mucosa.
T A B L E 1. S ~ Y OF HELICOBACTER PYLORI ANTIGENS B Y ~ E C U L A R WEIGHT D E ' ~ R M I N E D BY IMMUNOBLOT"
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Reactive proteins 011)a)
Specificity
Location
Reactivity
>180
Common
Soluble
Minor
180
Strain
100-130
Strain
Soluble
Strain-specific major band
Minor
93
Species
Major
86-88
Strain
Associated with 120 kDa?
75
Species
Major
67
Species
Major
60-63
Species
Surface
Major
54-59
Common
Flagellar
Cross-reactive
>30-79
Common
Cross-reactive
<30
Common
Cross-reactive
aRelativesizes t~orted for immunoblotreactive~
will differbecause of diffetan~..,~in ~ g e n extractiontechniques.
I
Clinical and Laboratory Diagnosis of H. pylori: Nonserologic Methods A number of methods for the diagnosis of H. pylori have been evaluated. 23~ Direct endoscopic biopsy of involved gastric mucosa, including the advantage of mucosal histopathologic assessment, is an invasive, expensive, but sensitive method of H. pylori diagnosis. Microbiologic culture of gastric mucosa is also a sensitive diagnostic method, of moderate expense, but features a substantial time delay to identify the organisms. Culture has the advantage of providing potential antibiotic susceptibility data, which is and will continue to be important in the treatment of the organism. Microscopic identification on histologic specimens is less sensitive and specific than culture. Although it is expensive and features a modest time delay, it allows the histological definition of the disease. 32In addition to hematoxylin and eosin, Gram stain, Giemsa stain, and a variety of silver stains have been used to observe the bacterium in tissue. An immunoperoxidase procedure featuring a monoclonal antibody is available but expensive and may be difficult to interpret. The detection of urease is a rapid and sensitive method of determining H. pylori presence on biopsy fragments removed at the time of gastroscopy.36The placement of the tissue specimen on urease agar has been standardized to a 95% sensitivity. After reading the agar reaction, the specimen can sub-
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sequently be submitted for histologic examination. The urea breath test is a highly specific and sensitive method of H. pylori detection that does not require biopsyfl This technique features the swallowing of radioactive urea with subsequent hydrolysis in the stomach and exhalation of radioactive carbon dioxide in an infected patient. Uninfected patients will clear the ingested radioactive urea in the urine. Although sensitive and specific, some laboratories find the handling of the radioactive specimens cumbersome and inconvenient.
Serological testing offers a fourth type of diagnostic assay that is attractive because it involves a relatively noninvasive procedure and may identify patients missed by testing a limited number of biopsy specimens. It appears useful as a screening procedure, and immunoblot analysis of specific surface antigens extracted from H. pyiori may prove important in resolving the status of potentially false-positive and false-negative results of other diagnostic tests. However, the reported results from serological testing have been conflicting. The discrepancies in previous studies can be explained by the variability in the techniques used to validate serological results, which include differences in antigen preparations used in the serological assays, cliniI II1_! ......... ©
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cal differences in the study populations. and the difficulty in making a diagnosis of H. pylori-assoeiated gastric fKsease, For example, Nedenskov-Sorensen et al. found that at least three biopsies were necessary for histological staining to approach the sensitivity of culture for diagnosis of H. pylori gastric disease and to ensure an adequate specimen for assessment of histological gastritis. 26Numerous other publications have addressed the problems associated with the abiSty to reliably culture H. pylori from biopsy tissue,zt'mm Even with these difficulties, serological tests for antibody to H. pytori approach a sensitivity and specificity in the 80%-90% range. 17'33A0The format for the assays also varies from laboratory-based EIA to rapid tests with latex particles coated with solubilized antigens for clinical use. EIA appears to be the most specific assay; rapid. serological assays offer enhanced sensitivity and the convenience of performance.
Serological Diagnosis of H. pyiori
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Aatigea Prqmratioas Antigen preparations for assays have utilized c e l l - s ~ Izroteins disrupted by sonication of whole cells, solubilized by acid extractions, or gently removed by suspension of the organism in distilled water.6a6~2Extracted surface proteins provide a greater specificity in serological assays than whole-cell sonicates, which I
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yield the greater sensitivity.~°'~2The suggestion is that whole-cell preparations have antigens common to other bacteria found on culture of gastric biopsies. Lipopolysaccharide (LPS) from H. pylori does exhibit a common species antigen and common antigens with other Gramnegative bacteria. However, LPS preparations varied in their reactivity with immune sera by immunoblot analysis and lacked sensitivity and specificity compared with solubilized surface proteins. Therefore, LPS has not been a good candidate antigen for serological tests. The O somatic antigens of LPS are swain specific and may prove useful in establishing a typing system for this organism 2A As has been reported, organisms other than H. pylori are isolated from 70% to 80% of gastric biopsy cultures.29'3°These organisms may contribute to gastritis either alone or in conjunction with H. pylori and, subsequently, to development of serum antibody. Campylobacter has flagellar antigens shown to crossreact with H. pylori and may therefore be a cause of false-positive serological test results. 2t In addition, rabbit antisera to Escherichia coli and Haemophilus influenzae have been shown to react with SDS extracts ofH. pyIori by immunoblot.3° The observation that antibody to H. pylori can crossreact with normal human gastric mucosal tissue is interesting from both a serologic and a pathologic viewpoint but may also contribute to false-positive reactions. 27In any case, antigen preparations from whole cells require further purification to enhance assay specificity without diminishing sensitivity. Table 1 summarizes the protein antigens of H. pylori as defined by immunoblots. False-negative serological tests are often attributed to an early infection when antibody has not yet been produced in significant quantity or to an anergic patient. Some serological tests use multiple classes of antibody to include IgA, IgM, and IgG to prevent false-negative reactions. IgAclass antibodies are in tissue secretions and can appear systemically. However, all IgG subclasses predominate as circulating antibodies without a significant IgM response. Accordingly, most false-negative serologi-
cal reactions may the result of antigenic differences between the antigen preparations used in assays and the H. pylori strain causing disease.
Immunobiot Analysis In our laboratory, immunoblot analysis of SDS extracts of 13 H. pylori isolates, using patients' sera, revealed an association
Given
the diversity of H. p y l o r i isolates from different patients and the possibility of significant antigenic variability, it seems reasonable to recommend that multiple H. p y l o r i strains be used for preparation of antigen for serological tests to optimize the detection of it.
between the presence of 97-120 kDa proteins and patients with acute, but not chronic, gastritis. The protein in this size range appears to correlate with the cytotoxin of H. pylori and may play a part in the type of gastric disease. 3,8,9 In addition, the presence of the high-molecular-weight protein band in immunoblots was associated with duodenal and peptic ulcers, t8'~9Crabtree et al. found an association between peptic ulceration and acute gastritis and 120-kDa producing strains ofH. pylori on IgA immunoblots.8 Using IgG and IgA immunoblots, Von Wulffen et al. reported good correlation between the presence of H. pylori and detection of certain protein bands, including a 110-kDa band. 38IgA antibody, however, was present at lower titers than IgG antibody, and IgM antibody was nonspecific. Kaldor et al. found detection of antibody to antigens in the 100-kDa region of ira-
© 1993 Elsevier Science Publishing Co., Inc.
munoblots to be the most reliable diagnostically. 18I-Iirschl et al. used the 120-kDa protein, which was thought to be the same as Kaldor's 100-kDa protein, in an F_LISA test and found high specificity (95%-98%) but low sensitivity (70%-84%)) 6 This high-molecular-weight H. pylori-specific band was only detected by sera from patients whose H. pylori isolates also expressed the protein. This suggests that the presence of this protein is variable in H. pylori isolates, and there may be antigenic differences in the high-molecular-weight proteins (>75 kDa) among producing strains. 35 In addition, a recently cloned gene from H. pylori (cagA), believed to be associated with the cytotoxic activity, exhibited restriction fragment polymorphisms between strains. 35Therefore, it is not clear to what extent the multiple bands in the >90-kDa region of immunoblots represent variations of a single protein family. Given the diversity of H. pylori isolates from different patients and the possibility of significant antigenic variability, it seems reasonable to recommend that multiple H. pylori strains be used for preparation of antigen for serological tests to optimize the detection of H. pylori.
Use of Serology and Immunoblots An example of the usefulness of serology in determining a diagnosis of H. pylori gastritis was evident in one of our patients with a histological diagnosis of chronic inflammation. The biopsies were negative by culture and direct urease assay. No organisms were seen during histological examination for H. pylori. The patient's serum was positive by two commercially available serological tests. Immunoblot examination was performed using the patient's serum and clinical H. pylori isolates from other patients. This detected weakly positive protein bands characteristic of H. pylori near the 97-, 66-, and <42.7-kDa markers. Therefore, this patient was positive by serology and had an immunoblot pattern consistent with a serological response to a previous infection with H. py-
lori. A second example was a patient with a histological diagnosis of acute gastritis and biopsies positive by culture and a di-
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128 CLINICAL I M M U N O L O G Y Newsletter
rect urease test. No H. pylori-like organisms were observed by histology, and serological tests were negative. Her serum was tested by immunoblot against whole-cell SDS extracts of her own and 6 H. pylori isolates from other patients. Bands at approximately 100, 84, and 64 kDa and many smaller protein bands of less than 60 kDa were detected by immunoblot using her serum as the primary antibody source against her own and 3 of the other 6 H. pylori isolates. Against the additional 3 H. pylori isolates, only a few low-molecularweight bands (<40 kDa) were reactive. This patient's serum was, therefore, a false negative by the commercially available serological tests used but was immunoblot positive. The failure to react against 3 of 7 H. pylori isolates by immunoblot points to the possible diversity of clinical isolates and therefore variability of antigen preparations of commercially available serological tests.
have a sensitivity and specificity that correlates with biopsy and culture results. In addition, immunoblot analysis of patients' sera to their H. pylori isolate is necessary in evaluating the serological response and may prove helpful in the diagnosis of the type of H. pylori-associated disease. References 1.
2.
3.
4. 5. 6.
Immunology and Disease Whether defining the immunological response of patients by their reaction to various antigens of H. pylori can determine the type of gastric disease remains to be pursued. Immune response of patients is most likely dependent upon their disease state. H. pylori can be a colonizer of the gaslric mucosa or contribute to disease, whether that is gastritis, ulcerations, or cancer. The response of IgG-class antibodies appears to be the most consistent, whereas IgA and IgM are variable, t9 Seropositivity markedly increases with the age of sampled populations in developed countries but appears to be significantly higher in pediatric populations in underdeveloped countries. These data may be suspect due to the degree of crossreacting gastrointestinal parasites (i.e., Campylobacter species) prevalent in these populations. ~What appears to be contermed is the ability of serology to monitor the response of treatment for H. pyiori infections, t3,2°In these studies, titers of immunoglobulin classes significantly decrease within 6 months of successful therapy. Overall, serological tests for the diagnosis of H. pylori-associated gastric disease II 0197-1859/93/$0.00 + 6.00
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7.
8.
9.
10.
11.
12.
13.
14.
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Anderson LP, Espersen F: Immunoglobulin G antibodies to Helicobacter pylori in patients with dyspeptic symptoms investigated by the Western immunoblot technique. J Clin Microbiol 30:1743-1751, 1992. Ansorg R, yon Recklinghansen G, Pomarius R, et al.: Evaluation of techniques for isolation, subcultivation, and preservation of Helicobacter py. Iori. J Clin Microbio129:51-53, 1991. Apel I, Jacobs E, Kist M, et al.: Antibody response of patients against a 120-kDa surface protein of Campylobacter pylori. Zbl Bakt Hyg A 268:271-276, 1988. Blaser M-J: Helicobacterpytori: Its role in disease. Clin Infec Dis 15:386-393, 1992. Correa P, Fox J, et al.: Helicobacter pylori and gastric carcinoma. Cancer 66:2569-2574, 1990. Cover TL, Dooley CP, Blaser M J: Characterization of and human serologic response to proteins in Helicobacter pylori broth culture supematants with vacuolizing cytotoxin activity. Infec Immnnol 58:603-610, 1990. Cover TL, Blaser M J: Helicobacter pylori and gastroduodenal disease. Am Rev Med 43:135145, 1992. Crabtree JE, Taylor JD, Wyatt Jl, et al.: Mucosal lgA recognition of Helicobacter pylori 120-kDa protein, peptic ulceration, and gastric pathology. Lancet 338:332-335, 1991. Crabtree JE, Fignra N, Taylor JD, et al.: Expression of 120-kilodalton protein and cytotoxicity in Helicobacter pylori. J Clin Pathol 45:733-734, 1992. Faulde M, Schroder JP, Sobe D: 1992. Serodiagnosis of Helicobacter pylori infections by detection of immunoglobulin G antibodies using an immunoblot technique and enzyme immunoassay. Eur J Clin Microbiol Infect Dis 11:589-594, 1992. Gents RM, Hamncr W, Graham DY: 1993. Gastric lymphoid follicules in Helicobacter pylori infection. Hum Pathol 24:577-583, 1993. Gerstenecker B, Eschweiler B, Vogele H, et al.: Serodiagnosls of Helicobacter pylori infections with an enzyme immtmoassay using the chromatographically purified 120-kilodalton protein. Fur J Clin Microbiol Infect Dis 11:595-601, 1992. Glupczynski Y, Burette A, Goossens H, et al.: Effect of antimicrobial therapy on the specific serological response to Helicobacter pylori infection. Eur J Clin Microbiol Infect Dis 11:583-588, 1992. Glupczynski Y, Labbe M, Thibanmont F: 1989.
I
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Comparative evaluation of a new selective culture medium for improved isolation of Campylobacter pylori from gastric biopsy specimens~ In: Megraud F, Lamouliatte H (eds): Gastroduodenat Pathology and Campylobacter pylori. New York, Elsevier Science Publishers, 1989, Lap. 3--6. 15. Goodwin CS, Armstrong JA, Peters M: Microbiology of C. pylori. In: Blaser MJ (ed): Campylobacter pylori Gastritis and Peptic Ulcer Disease. New York, lgaku-Shoin, 1989, pp. 25-51, 16. Rirschl AM, Rathbone BJ, Wyatt JL et aL: Comparison of ELISA antigen preparations alone or in combination for serodiagnusin8 Helicobacter pylori infections. J Clin Pathol 43:511-5t3, 1990. 17. Hoek FJ, Noach LA, Ranws EAJ, et al.: Evaluation of the performance of commercial test kits for detection of Helicobacter pylori antibodies ha serum. J Clin Microbiol 30:1525-1528, 1992. 18. Kaldor J, Tee W, Nicolacopolous C, et al.: lm~ munoblot confirmation of immune response to Campylobacter pyloridis in patients with duode.. nal ulcers. Med J Aust 145:133-135, 1986, 19. KistM: ImmunologyofHelicobacterpylori. In: Marshall B J, McCallum RW, Gwerrant RL, et al. (eds): Helicobacterpylori in Peptic Ulceration and Gastritis. Boston, Blackwell Scientific, 1991, pp. 92-..110. 20. Kosnnen TU, Seppala K, Sama S, et al.: Diago nostic value of decreasing lgG, [gA and lgM antibody titers after eradication of Helicobacter pylori. Lancet 339:893-895, 1992. 21. Lee A, Logan SM, Trust TJ: Demonstration of a flagellar antigen shared by a diverse group of spiral-shaped bacteria that colonize intestinal mucus. Infect Immunol 55:828-831, 1987 22. Lelwala-Gumqe J, Nilsson I, Ljunqh A, et al.: Cell surface proteins of Helicobacter pylori as antigens in an ELISA and comparison with three commercial ELISA. Scand J Infect Dis 24:457465, 1992. 23. Marshall B J: Practical diagnosis of Helicobacter pylori. In: Marshall BJ, McCallum RW, Gwerrant RL, et al. (eds): Helicobacter pylori in Peptic Ulceration and Gastritis. Boston, Blackwell Scientific., 1991. pp. 139-159. 24. Mills SD, Kurjanczyk LA, Penner JL: Antigenicity of Helicobacter pylori lipopoly saccharides. J Clin Microbiol 30:3175-3180, 1992. 25. Mobley HLT, Cortesia MJ, Rosenthat LE, et al.: Characterization of urease from Campylobacter pylori. J Clin Microbiol 26i831-836; 1988. 26. Nedenskov-Sorensen P, Aase S, Bjomeklett A. et al.: Sampling efficiency in the diagnosis of ttelicobacter pylori infection and chronic active gastritis. J Clin Microbiol 29:672-675,1991. 27. Negrini R, Lisato L, Zanella I, et al.: Helicobacterpylori infection induces antibodies cross-reacting with human gastric mucosa. Gastroenterology 101:437--445, 1991. 28. Nichols L, Sugbaoier M, et al.: Evaluation of diagnostic methods for Helicobacter pylori gastritis. Am J Clin Pathol 95:769-773, 1991. I I
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29. Schrader JA, Peck HV, Notis WM, et al.: A role for culture in diagnosis of Helicobacter pylori related gastric disease. Am J Gastroenterol, in press. 30. Sjnstedt S, Kager L, Veress B, et al.: Campylobacter pylori in relation to other microorganisms on gastric mucosa. In: Megraud F, Lamouliatte H (eds): Gastroduodenal Pathology and Campylobacter pylori. New York, Elsevier Science Publishers, 1989, pp. 35-38.
33.
34.
35.
31. Soltesz V, Zeeberg B, Wadstrom T: 1992. Optimal survival of Helicobacter pylori under various transport conditions. Clin Microbiol 30:1453-1456, 1992. 32. Strickland RG, Fenoglio-Preiser CM: 1991 Gastritis: Classification and histology then and now. In: Marshall BJ, McCallum RW, Gwerrant RL, et al. (eds): Helicobacter pylori in Peptic Ulcera-
36.
37.
tion and Gastritis. Boston, Blackwell Scientific, 1991, pp. 1-18. Talley NJ, Kost LA, Haddad AR, et al.: Comparison of commercial serological tests for detection of Helicobacter pylori antibodies. JCM 30:31463150, 1992. Taylor DN, Blaser M J: The epidemiology of Helicobacter pylori infection. Epidemiol Rev 13:42-59, 1992. Tummum NKR, Cover TL, Blaser M J: Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: Evidence of linkage to cytotoxin production. Infec Immunol 61:1799-1809, 1993. Vaira D, Holton J, Calms S, et al.: Urease tests for Campylobacter pylori: Care in interpretation. J Clin Pathol 41:812---813, 1988. Vandenplas Y, Blecker U, Devreker T, et al.:
Contribution of the
13C-ureabreath test to the de-
tection of Helicobacterpylori gastritis in chil-
dren. Pediatrics 90:608-611, 1992. 38. Von Wulffen H, Grote ILl, Gterman S, et al.: Immunoblot analysis of immune response to Campylobacter pylori and its clinical associations. J Clin Pathol 41:653--659, 1988. 39. Warren JR, Marshall BJ: Unidentified curved baccilli on gastric epithelium in chronic active gastritis. Lancet 1:1273-1275, 1983. 40. Westblom TU, Madan E, Gudipati S, et al.: Diagnosis of Helicobacter pylori infection in adult and pediatric patients by using Pyloriset, a rapid latex agglutination test. J Clin Microbiol 30:9698, 1992.
Vaccination Against Diarrheal Disease Thomas L. Hale Walter Reed Army Institute of Research, Washington, D.C.
n a global basis, infectious diarrheal disease is estimated to cause up to 5 million deaths annually, exceeding pneumonia and tuberculosis as the most deadly of infectious diseases. In addition to the occasional fatal infection, debilitating diarrheal episodes average up to 10 per year during the first 2 years of life in many areas of Africa, Latin America, Asia, and Oceania. Even adults living in endemic locales can be stricken with serious or fatal infections during epidemics of Vibrio cholerae or Shigella dysenteriae type 1,1 With approximately 20 million travelers from industrialized countries visiting developing countries every year, the 30%-50% diarrheal attack rate in these immunologically naive visitors has a serious impact on health-care utilization in both the developing countries and the countries of origin.2 Thus, diarrheal disease impedes both individual and national development while extracting a huge cost in treatment and control programs. The obvious public-
O
health solutions of reticulated water and safe sewage disposal are utopian considering the daunting costs and the indifference of industrialized countries. Administration of vaccines that alleviate diarrheal symptoms in native children and in foreign visitors remains the most realistic current approach. Endemic diarrheal disease in nonindustrialized countries is associated with rotavirus, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli, Shigella flexneri, Campylobacter jejuni, and nontyphoidal Salmonella species, typically in descending order of prevalence. However, epidemiological surveys of diarrheal etiology in household settings are complicated by a substantial incidence of putative enteric pathogens isolated from apparently healthy individuals. For example, only about one of three infections with ETEC and one in four infections with V. cholerae E1 Tor are associated with disease. 2 These observations suggest that nonspecific host© 1993 Elsevier Science Publishing Co., Inc.
defense mechanisms, or even actively acquired immunity to commonly encountered intestinal pathogens, does not necessarily prevent infection but rather reduces the infectious dose to a subclinical level. In this balance, the total number of infectious organisms that survive gastric transit and arrive at the intestinal niche is a key factor in determining the clinical manifestation of the infection (i.e., subclinical colonization or diarrhea). Thus, the most realistic goal of vaccination is to reduce the infectious load of a diarrheal agent to a subclinical level. Intuition and experience suggest that the difficulty of achieving this goal is a function of the minimum inoculum size that elicits clinical disease in a substantial proportion of individuals. For example, the infective dose of V. cholera or ETEC in volunteers is in 106~ colonyforming units (CFU), 3-s and immune protection against these noninvasive organisms is more readily achieved than is protection against enteroinvasive Shigella 0197-1859/93/$0.00 + 6.00
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phia, JB Lippincott, 1988, pp. 692-700. 34. Rump JA, Woemer I, Roth M, et al.: A new type of ANCA in active ulcerative colitis: Correlation to therapy and disease activity. In: Gross WL (ed): Advances in Experimental Medicine and Biology. Abstract Book of the 4th International Workshop on ANCA. London, Plenum, 1992, p. 36. 35. Sachar DB: Ulcerative colitis and sclerosing cholangitis: Does "IBD" mean "Inflamed Bile Ducts"? (editorial). Gastroenterology 100:14691470, 1991. 36. Saxon A, Shanahan F, Landers C, et al.: A distinct subset of antineutrophil cytoplasmic antibodies is associated with inflammatory bowel
37.
38.
39.
40.
disease. J Allergy Clin Immunol 86:202-210, 1990. Seibold F, Weber P, Klein R, et al.: Clinical significance of antibodies against neutrophils in patients with inflanunatory bowel disease and primary sclerosing cholangitis. Gut 33:657-662, 1992. Shanahan F, Duerr R, Rotter Yl, et al.: Neutrophil autoantibodies in ulcerative colitis: Familial aggregation and genetic heterogeneity. Gastroenterology 103:456-461, 1992. Shanahan F, Landers C, Duerr R: Neutrophil autoantibodies as disease markers for ulcerative colitis. Immunol Res 10:479-484, 1991. Snook JA, Chapman RW, Fleming K, et al.: Anti-
neutrophil nuclear antibody in ulcerative colitis, Crohn's disease and primary sclerosing cholangitis. Clin Exp Inununol 76:30--33, 1989. 41. Wiik A: Delineation of a standard method for indirect immunofluorescence detection of ANCA. Acta Pathol Microbiol Scand 97(Suppl 6):12-13, 1989. 42, Zauli D, Baffoni L, Cassani F, et al.: Antineutrophil cytoplasmic antibodies in primary sclerosing cholangitis, ulcerative colitis, and autoinunune diseases (letter). Gastroenterology 102:10881089, 1992.
Serologic Diagnosis of H e l i c o b a c t e r pylori-Associated Gastrointestinal Disease Richard A. Venezia and Jeffrey S. Ross Department of Pathology and Laboratory Medicine, Albany Medical Center, Albany, New York
H
"elicobacter pylori causes a slow,
human gastroduodenal infection ,with a high infection rate that is asymptomatic in many patients. Gastric bacteria were first described in 1874, and over the next century endogenous human gastric urease activity was found to be associated with spiral-shaped intragastric organisms. A spiral bacterium was isolated and cultured in 1983 from the human stomach by Warren and Marshall. 39 Since then, a considerable volume of literature as to the nature of the organism and its potential role in human disease has been published. H. pylori has been isolated throughout the world. The normal prevalence rate varies from 0% to nearly 50% in any geographic site and socioeconomic status. 34In the United States, H. pylori prevalence during the first four decades of life increases from 0% to 10% and reaches 50% beyond the fifth and sixth decades. In Third World countries, a 10% prevalence in the first
decade of life many reach 50% in the second decade and 75% by the fourth decade. Microbiologic Features H. pylori is a Gram-negative coccobacillary to spiral-shaped bacterium with four to six unipolar sheathed flagellae. It is fastidious, requiring a warm microaerobic environment, and has a strong affinity for human gastric-type epithelium. 15H. pylori may be isolated from the human stomach, containing normal or increased acid content, relatively easily using a variety of liquid and solid media featuring antibiotic supplements to control growth of competing organismsY 4,29,3~In the achlorhydric specimen, special techniques may be necessary as organisms other than H. pylori may colonize the human stomach in these conditions. The most characteristic biochemical property of H. pylori is urease production, which distinguishes it from the Campylobacter species and other related bacteria, z5
© 1993 Elsevier Science Publishing Co., Inc.
Although the 35-38 M percent guanosineplus-cytosine value for H. pylori places the organism in the range of the Campylobacter species, H. pylori is separated from Campylobacter by urease production, ultrastructural features, and fatty acid composition. The rRNA sequences of H. pylori are also different from Campylobacter as defined from the original Australian isolates and therefore warrants placement of this bacterium in the Helicobacter genus.
Human Gastrointestinal Disease Associated with H. pylori H. pylori has been isolated from normal human gastric antrum, duodenal metaplasia, and Meckel's diverticulum, but rarely from the rectum.4,7,32The organism has been isolated from 83% of patients with chronic superficial gastritis, 92% of patients with duodenal ulcer, 69% of patients with gastric ulcers, and 51% of patients with nonulcer dyspepsia. The organism has not been isolated generally
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126 CLINICAL I M M U N O L O G Y Newsletter
from esophageal disorders, nor is it identified in most cases of alrophic gastritis or nonsteroidal antiinflammatory drug-related gastritis. Relapse rates in duodenal ulcers have shown that H. pylori positive patients suffer relapse in 75%--95% of cases as compared with H. pylori negative patients with a less than 25% duodenal ulcer relapse rate. H. pylori has been linked in several major epidemiologic studies to the risk of gastric adenocarcinoma and lymphoma. TM The odds ratio of gastric cancer has ranged from 2.7 to 6.0. However, experimental animal and prospective clinical studies have not, as yet, defined a cause-effect relationship between the organism and precancerous and cancerous changes in the gastric mucosa.
T A B L E 1. S ~ Y OF HELICOBACTER PYLORI ANTIGENS B Y ~ E C U L A R WEIGHT D E ' ~ R M I N E D BY IMMUNOBLOT"
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Reactive proteins 011)a)
Specificity
Location
Reactivity
>180
Common
Soluble
Minor
180
Strain
100-130
Strain
Soluble
Strain-specific major band
Minor
93
Species
Major
86-88
Strain
Associated with 120 kDa?
75
Species
Major
67
Species
Major
60-63
Species
Surface
Major
54-59
Common
Flagellar
Cross-reactive
>30-79
Common
Cross-reactive
<30
Common
Cross-reactive
aRelativesizes t~orted for immunoblotreactive~
will differbecause of diffetan~..,~in ~ g e n extractiontechniques.
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Clinical and Laboratory Diagnosis of H. pylori: Nonserologic Methods A number of methods for the diagnosis of H. pylori have been evaluated. 23~ Direct endoscopic biopsy of involved gastric mucosa, including the advantage of mucosal histopathologic assessment, is an invasive, expensive, but sensitive method of H. pylori diagnosis. Microbiologic culture of gastric mucosa is also a sensitive diagnostic method, of moderate expense, but features a substantial time delay to identify the organisms. Culture has the advantage of providing potential antibiotic susceptibility data, which is and will continue to be important in the treatment of the organism. Microscopic identification on histologic specimens is less sensitive and specific than culture. Although it is expensive and features a modest time delay, it allows the histological definition of the disease. 32In addition to hematoxylin and eosin, Gram stain, Giemsa stain, and a variety of silver stains have been used to observe the bacterium in tissue. An immunoperoxidase procedure featuring a monoclonal antibody is available but expensive and may be difficult to interpret. The detection of urease is a rapid and sensitive method of determining H. pylori presence on biopsy fragments removed at the time of gastroscopy.36The placement of the tissue specimen on urease agar has been standardized to a 95% sensitivity. After reading the agar reaction, the specimen can sub-
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sequently be submitted for histologic examination. The urea breath test is a highly specific and sensitive method of H. pylori detection that does not require biopsyfl This technique features the swallowing of radioactive urea with subsequent hydrolysis in the stomach and exhalation of radioactive carbon dioxide in an infected patient. Uninfected patients will clear the ingested radioactive urea in the urine. Although sensitive and specific, some laboratories find the handling of the radioactive specimens cumbersome and inconvenient.
Serological testing offers a fourth type of diagnostic assay that is attractive because it involves a relatively noninvasive procedure and may identify patients missed by testing a limited number of biopsy specimens. It appears useful as a screening procedure, and immunoblot analysis of specific surface antigens extracted from H. pyiori may prove important in resolving the status of potentially false-positive and false-negative results of other diagnostic tests. However, the reported results from serological testing have been conflicting. The discrepancies in previous studies can be explained by the variability in the techniques used to validate serological results, which include differences in antigen preparations used in the serological assays, cliniI II1_! ......... ©
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cal differences in the study populations. and the difficulty in making a diagnosis of H. pylori-assoeiated gastric fKsease, For example, Nedenskov-Sorensen et al. found that at least three biopsies were necessary for histological staining to approach the sensitivity of culture for diagnosis of H. pylori gastric disease and to ensure an adequate specimen for assessment of histological gastritis. 26Numerous other publications have addressed the problems associated with the abiSty to reliably culture H. pylori from biopsy tissue,zt'mm Even with these difficulties, serological tests for antibody to H. pytori approach a sensitivity and specificity in the 80%-90% range. 17'33A0The format for the assays also varies from laboratory-based EIA to rapid tests with latex particles coated with solubilized antigens for clinical use. EIA appears to be the most specific assay; rapid. serological assays offer enhanced sensitivity and the convenience of performance.
Serological Diagnosis of H. pyiori
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Aatigea Prqmratioas Antigen preparations for assays have utilized c e l l - s ~ Izroteins disrupted by sonication of whole cells, solubilized by acid extractions, or gently removed by suspension of the organism in distilled water.6a6~2Extracted surface proteins provide a greater specificity in serological assays than whole-cell sonicates, which I
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Vol. 13, No. 9/10, 1993
yield the greater sensitivity.~°'~2The suggestion is that whole-cell preparations have antigens common to other bacteria found on culture of gastric biopsies. Lipopolysaccharide (LPS) from H. pylori does exhibit a common species antigen and common antigens with other Gramnegative bacteria. However, LPS preparations varied in their reactivity with immune sera by immunoblot analysis and lacked sensitivity and specificity compared with solubilized surface proteins. Therefore, LPS has not been a good candidate antigen for serological tests. The O somatic antigens of LPS are swain specific and may prove useful in establishing a typing system for this organism 2A As has been reported, organisms other than H. pylori are isolated from 70% to 80% of gastric biopsy cultures.29'3°These organisms may contribute to gastritis either alone or in conjunction with H. pylori and, subsequently, to development of serum antibody. Campylobacter has flagellar antigens shown to crossreact with H. pylori and may therefore be a cause of false-positive serological test results. 2t In addition, rabbit antisera to Escherichia coli and Haemophilus influenzae have been shown to react with SDS extracts ofH. pyIori by immunoblot.3° The observation that antibody to H. pylori can crossreact with normal human gastric mucosal tissue is interesting from both a serologic and a pathologic viewpoint but may also contribute to false-positive reactions. 27In any case, antigen preparations from whole cells require further purification to enhance assay specificity without diminishing sensitivity. Table 1 summarizes the protein antigens of H. pylori as defined by immunoblots. False-negative serological tests are often attributed to an early infection when antibody has not yet been produced in significant quantity or to an anergic patient. Some serological tests use multiple classes of antibody to include IgA, IgM, and IgG to prevent false-negative reactions. IgAclass antibodies are in tissue secretions and can appear systemically. However, all IgG subclasses predominate as circulating antibodies without a significant IgM response. Accordingly, most false-negative serologi-
cal reactions may the result of antigenic differences between the antigen preparations used in assays and the H. pylori strain causing disease.
Immunobiot Analysis In our laboratory, immunoblot analysis of SDS extracts of 13 H. pylori isolates, using patients' sera, revealed an association
Given
the diversity of H. p y l o r i isolates from different patients and the possibility of significant antigenic variability, it seems reasonable to recommend that multiple H. p y l o r i strains be used for preparation of antigen for serological tests to optimize the detection of it.
between the presence of 97-120 kDa proteins and patients with acute, but not chronic, gastritis. The protein in this size range appears to correlate with the cytotoxin of H. pylori and may play a part in the type of gastric disease. 3,8,9 In addition, the presence of the high-molecular-weight protein band in immunoblots was associated with duodenal and peptic ulcers, t8'~9Crabtree et al. found an association between peptic ulceration and acute gastritis and 120-kDa producing strains ofH. pylori on IgA immunoblots.8 Using IgG and IgA immunoblots, Von Wulffen et al. reported good correlation between the presence of H. pylori and detection of certain protein bands, including a 110-kDa band. 38IgA antibody, however, was present at lower titers than IgG antibody, and IgM antibody was nonspecific. Kaldor et al. found detection of antibody to antigens in the 100-kDa region of ira-
© 1993 Elsevier Science Publishing Co., Inc.
munoblots to be the most reliable diagnostically. 18I-Iirschl et al. used the 120-kDa protein, which was thought to be the same as Kaldor's 100-kDa protein, in an F_LISA test and found high specificity (95%-98%) but low sensitivity (70%-84%)) 6 This high-molecular-weight H. pylori-specific band was only detected by sera from patients whose H. pylori isolates also expressed the protein. This suggests that the presence of this protein is variable in H. pylori isolates, and there may be antigenic differences in the high-molecular-weight proteins (>75 kDa) among producing strains. 35 In addition, a recently cloned gene from H. pylori (cagA), believed to be associated with the cytotoxic activity, exhibited restriction fragment polymorphisms between strains. 35Therefore, it is not clear to what extent the multiple bands in the >90-kDa region of immunoblots represent variations of a single protein family. Given the diversity of H. pylori isolates from different patients and the possibility of significant antigenic variability, it seems reasonable to recommend that multiple H. pylori strains be used for preparation of antigen for serological tests to optimize the detection of H. pylori.
Use of Serology and Immunoblots An example of the usefulness of serology in determining a diagnosis of H. pylori gastritis was evident in one of our patients with a histological diagnosis of chronic inflammation. The biopsies were negative by culture and direct urease assay. No organisms were seen during histological examination for H. pylori. The patient's serum was positive by two commercially available serological tests. Immunoblot examination was performed using the patient's serum and clinical H. pylori isolates from other patients. This detected weakly positive protein bands characteristic of H. pylori near the 97-, 66-, and <42.7-kDa markers. Therefore, this patient was positive by serology and had an immunoblot pattern consistent with a serological response to a previous infection with H. py-
lori. A second example was a patient with a histological diagnosis of acute gastritis and biopsies positive by culture and a di-
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128 CLINICAL I M M U N O L O G Y Newsletter
rect urease test. No H. pylori-like organisms were observed by histology, and serological tests were negative. Her serum was tested by immunoblot against whole-cell SDS extracts of her own and 6 H. pylori isolates from other patients. Bands at approximately 100, 84, and 64 kDa and many smaller protein bands of less than 60 kDa were detected by immunoblot using her serum as the primary antibody source against her own and 3 of the other 6 H. pylori isolates. Against the additional 3 H. pylori isolates, only a few low-molecularweight bands (<40 kDa) were reactive. This patient's serum was, therefore, a false negative by the commercially available serological tests used but was immunoblot positive. The failure to react against 3 of 7 H. pylori isolates by immunoblot points to the possible diversity of clinical isolates and therefore variability of antigen preparations of commercially available serological tests.
have a sensitivity and specificity that correlates with biopsy and culture results. In addition, immunoblot analysis of patients' sera to their H. pylori isolate is necessary in evaluating the serological response and may prove helpful in the diagnosis of the type of H. pylori-associated disease. References 1.
2.
3.
4. 5. 6.
Immunology and Disease Whether defining the immunological response of patients by their reaction to various antigens of H. pylori can determine the type of gastric disease remains to be pursued. Immune response of patients is most likely dependent upon their disease state. H. pylori can be a colonizer of the gaslric mucosa or contribute to disease, whether that is gastritis, ulcerations, or cancer. The response of IgG-class antibodies appears to be the most consistent, whereas IgA and IgM are variable, t9 Seropositivity markedly increases with the age of sampled populations in developed countries but appears to be significantly higher in pediatric populations in underdeveloped countries. These data may be suspect due to the degree of crossreacting gastrointestinal parasites (i.e., Campylobacter species) prevalent in these populations. ~What appears to be contermed is the ability of serology to monitor the response of treatment for H. pyiori infections, t3,2°In these studies, titers of immunoglobulin classes significantly decrease within 6 months of successful therapy. Overall, serological tests for the diagnosis of H. pylori-associated gastric disease II 0197-1859/93/$0.00 + 6.00
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7.
8.
9.
10.
11.
12.
13.
14.
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Anderson LP, Espersen F: Immunoglobulin G antibodies to Helicobacter pylori in patients with dyspeptic symptoms investigated by the Western immunoblot technique. J Clin Microbiol 30:1743-1751, 1992. Ansorg R, yon Recklinghansen G, Pomarius R, et al.: Evaluation of techniques for isolation, subcultivation, and preservation of Helicobacter py. Iori. J Clin Microbio129:51-53, 1991. Apel I, Jacobs E, Kist M, et al.: Antibody response of patients against a 120-kDa surface protein of Campylobacter pylori. Zbl Bakt Hyg A 268:271-276, 1988. Blaser M-J: Helicobacterpytori: Its role in disease. Clin Infec Dis 15:386-393, 1992. Correa P, Fox J, et al.: Helicobacter pylori and gastric carcinoma. Cancer 66:2569-2574, 1990. Cover TL, Dooley CP, Blaser M J: Characterization of and human serologic response to proteins in Helicobacter pylori broth culture supematants with vacuolizing cytotoxin activity. Infec Immnnol 58:603-610, 1990. Cover TL, Blaser M J: Helicobacter pylori and gastroduodenal disease. Am Rev Med 43:135145, 1992. Crabtree JE, Taylor JD, Wyatt Jl, et al.: Mucosal lgA recognition of Helicobacter pylori 120-kDa protein, peptic ulceration, and gastric pathology. Lancet 338:332-335, 1991. Crabtree JE, Fignra N, Taylor JD, et al.: Expression of 120-kilodalton protein and cytotoxicity in Helicobacter pylori. J Clin Pathol 45:733-734, 1992. Faulde M, Schroder JP, Sobe D: 1992. Serodiagnosis of Helicobacter pylori infections by detection of immunoglobulin G antibodies using an immunoblot technique and enzyme immunoassay. Eur J Clin Microbiol Infect Dis 11:589-594, 1992. Gents RM, Hamncr W, Graham DY: 1993. Gastric lymphoid follicules in Helicobacter pylori infection. Hum Pathol 24:577-583, 1993. Gerstenecker B, Eschweiler B, Vogele H, et al.: Serodiagnosls of Helicobacter pylori infections with an enzyme immtmoassay using the chromatographically purified 120-kilodalton protein. Fur J Clin Microbiol Infect Dis 11:595-601, 1992. Glupczynski Y, Burette A, Goossens H, et al.: Effect of antimicrobial therapy on the specific serological response to Helicobacter pylori infection. Eur J Clin Microbiol Infect Dis 11:583-588, 1992. Glupczynski Y, Labbe M, Thibanmont F: 1989.
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Comparative evaluation of a new selective culture medium for improved isolation of Campylobacter pylori from gastric biopsy specimens~ In: Megraud F, Lamouliatte H (eds): Gastroduodenat Pathology and Campylobacter pylori. New York, Elsevier Science Publishers, 1989, Lap. 3--6. 15. Goodwin CS, Armstrong JA, Peters M: Microbiology of C. pylori. In: Blaser MJ (ed): Campylobacter pylori Gastritis and Peptic Ulcer Disease. New York, lgaku-Shoin, 1989, pp. 25-51, 16. Rirschl AM, Rathbone BJ, Wyatt JL et aL: Comparison of ELISA antigen preparations alone or in combination for serodiagnusin8 Helicobacter pylori infections. J Clin Pathol 43:511-5t3, 1990. 17. Hoek FJ, Noach LA, Ranws EAJ, et al.: Evaluation of the performance of commercial test kits for detection of Helicobacter pylori antibodies ha serum. J Clin Microbiol 30:1525-1528, 1992. 18. Kaldor J, Tee W, Nicolacopolous C, et al.: lm~ munoblot confirmation of immune response to Campylobacter pyloridis in patients with duode.. nal ulcers. Med J Aust 145:133-135, 1986, 19. KistM: ImmunologyofHelicobacterpylori. In: Marshall B J, McCallum RW, Gwerrant RL, et al. (eds): Helicobacterpylori in Peptic Ulceration and Gastritis. Boston, Blackwell Scientific, 1991, pp. 92-..110. 20. Kosnnen TU, Seppala K, Sama S, et al.: Diago nostic value of decreasing lgG, [gA and lgM antibody titers after eradication of Helicobacter pylori. Lancet 339:893-895, 1992. 21. Lee A, Logan SM, Trust TJ: Demonstration of a flagellar antigen shared by a diverse group of spiral-shaped bacteria that colonize intestinal mucus. Infect Immunol 55:828-831, 1987 22. Lelwala-Gumqe J, Nilsson I, Ljunqh A, et al.: Cell surface proteins of Helicobacter pylori as antigens in an ELISA and comparison with three commercial ELISA. Scand J Infect Dis 24:457465, 1992. 23. Marshall B J: Practical diagnosis of Helicobacter pylori. In: Marshall BJ, McCallum RW, Gwerrant RL, et al. (eds): Helicobacter pylori in Peptic Ulceration and Gastritis. Boston, Blackwell Scientific., 1991. pp. 139-159. 24. Mills SD, Kurjanczyk LA, Penner JL: Antigenicity of Helicobacter pylori lipopoly saccharides. J Clin Microbiol 30:3175-3180, 1992. 25. Mobley HLT, Cortesia MJ, Rosenthat LE, et al.: Characterization of urease from Campylobacter pylori. J Clin Microbiol 26i831-836; 1988. 26. Nedenskov-Sorensen P, Aase S, Bjomeklett A. et al.: Sampling efficiency in the diagnosis of ttelicobacter pylori infection and chronic active gastritis. J Clin Microbiol 29:672-675,1991. 27. Negrini R, Lisato L, Zanella I, et al.: Helicobacterpylori infection induces antibodies cross-reacting with human gastric mucosa. Gastroenterology 101:437--445, 1991. 28. Nichols L, Sugbaoier M, et al.: Evaluation of diagnostic methods for Helicobacter pylori gastritis. Am J Clin Pathol 95:769-773, 1991. I I
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29. Schrader JA, Peck HV, Notis WM, et al.: A role for culture in diagnosis of Helicobacter pylori related gastric disease. Am J Gastroenterol, in press. 30. Sjnstedt S, Kager L, Veress B, et al.: Campylobacter pylori in relation to other microorganisms on gastric mucosa. In: Megraud F, Lamouliatte H (eds): Gastroduodenal Pathology and Campylobacter pylori. New York, Elsevier Science Publishers, 1989, pp. 35-38.
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31. Soltesz V, Zeeberg B, Wadstrom T: 1992. Optimal survival of Helicobacter pylori under various transport conditions. Clin Microbiol 30:1453-1456, 1992. 32. Strickland RG, Fenoglio-Preiser CM: 1991 Gastritis: Classification and histology then and now. In: Marshall BJ, McCallum RW, Gwerrant RL, et al. (eds): Helicobacter pylori in Peptic Ulcera-
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tion and Gastritis. Boston, Blackwell Scientific, 1991, pp. 1-18. Talley NJ, Kost LA, Haddad AR, et al.: Comparison of commercial serological tests for detection of Helicobacter pylori antibodies. JCM 30:31463150, 1992. Taylor DN, Blaser M J: The epidemiology of Helicobacter pylori infection. Epidemiol Rev 13:42-59, 1992. Tummum NKR, Cover TL, Blaser M J: Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: Evidence of linkage to cytotoxin production. Infec Immunol 61:1799-1809, 1993. Vaira D, Holton J, Calms S, et al.: Urease tests for Campylobacter pylori: Care in interpretation. J Clin Pathol 41:812---813, 1988. Vandenplas Y, Blecker U, Devreker T, et al.:
Contribution of the
13C-ureabreath test to the de-
tection of Helicobacterpylori gastritis in chil-
dren. Pediatrics 90:608-611, 1992. 38. Von Wulffen H, Grote ILl, Gterman S, et al.: Immunoblot analysis of immune response to Campylobacter pylori and its clinical associations. J Clin Pathol 41:653--659, 1988. 39. Warren JR, Marshall BJ: Unidentified curved baccilli on gastric epithelium in chronic active gastritis. Lancet 1:1273-1275, 1983. 40. Westblom TU, Madan E, Gudipati S, et al.: Diagnosis of Helicobacter pylori infection in adult and pediatric patients by using Pyloriset, a rapid latex agglutination test. J Clin Microbiol 30:9698, 1992.
Vaccination Against Diarrheal Disease Thomas L. Hale Walter Reed Army Institute of Research, Washington, D.C.
n a global basis, infectious diarrheal disease is estimated to cause up to 5 million deaths annually, exceeding pneumonia and tuberculosis as the most deadly of infectious diseases. In addition to the occasional fatal infection, debilitating diarrheal episodes average up to 10 per year during the first 2 years of life in many areas of Africa, Latin America, Asia, and Oceania. Even adults living in endemic locales can be stricken with serious or fatal infections during epidemics of Vibrio cholerae or Shigella dysenteriae type 1,1 With approximately 20 million travelers from industrialized countries visiting developing countries every year, the 30%-50% diarrheal attack rate in these immunologically naive visitors has a serious impact on health-care utilization in both the developing countries and the countries of origin.2 Thus, diarrheal disease impedes both individual and national development while extracting a huge cost in treatment and control programs. The obvious public-
O
health solutions of reticulated water and safe sewage disposal are utopian considering the daunting costs and the indifference of industrialized countries. Administration of vaccines that alleviate diarrheal symptoms in native children and in foreign visitors remains the most realistic current approach. Endemic diarrheal disease in nonindustrialized countries is associated with rotavirus, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli, Shigella flexneri, Campylobacter jejuni, and nontyphoidal Salmonella species, typically in descending order of prevalence. However, epidemiological surveys of diarrheal etiology in household settings are complicated by a substantial incidence of putative enteric pathogens isolated from apparently healthy individuals. For example, only about one of three infections with ETEC and one in four infections with V. cholerae E1 Tor are associated with disease. 2 These observations suggest that nonspecific host© 1993 Elsevier Science Publishing Co., Inc.
defense mechanisms, or even actively acquired immunity to commonly encountered intestinal pathogens, does not necessarily prevent infection but rather reduces the infectious dose to a subclinical level. In this balance, the total number of infectious organisms that survive gastric transit and arrive at the intestinal niche is a key factor in determining the clinical manifestation of the infection (i.e., subclinical colonization or diarrhea). Thus, the most realistic goal of vaccination is to reduce the infectious load of a diarrheal agent to a subclinical level. Intuition and experience suggest that the difficulty of achieving this goal is a function of the minimum inoculum size that elicits clinical disease in a substantial proportion of individuals. For example, the infective dose of V. cholera or ETEC in volunteers is in 106~ colonyforming units (CFU), 3-s and immune protection against these noninvasive organisms is more readily achieved than is protection against enteroinvasive Shigella 0197-1859/93/$0.00 + 6.00