Helicobacter (Campylobacter) pylori: a new twist to an old disease.

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HELIICOBACTER (CAMPYLOBACTER) PYL()RI: A New Twist to an Old Disease James D. Dick Department of Laboratory Medicine, The Johns Hopkins Medical Institutions, Balti more, Maryland 21205 KEY WORDS:

gastritis, peptic ulcer, virulence factors, diagnosis

CONTENTS INTRODUCTION.....................................................................................

249

BACTERIOLOGY OF CAMPYLOBACTER pyLORI....................... ...................

250 250 250 253 255 255 259 260 261 261 262 262 263 263

Morphological Characteristics..... . . ....... . . . . . ...... . . ....... . . . .............. . . . ........... Metabolism and Physiology........ . . ......... . .. ....... ................ . . ... ................... Potential Virulence Factors..................................................................... DIAGNOSIS ....... ... ......... . ............ ........... ....... .... .......... .... .... ......... .......... Gastric Biopsy..................................................................................... Serology ......... ............ ........... ............... ...... ............ ......... ......... ......... Urea Breath Test . . ........ . . . . ......... . . . ....... ......... .................. . .............. . .... Nucleic Acid Hybridization and Antigen Detection Methods ....... . .................. .

. . .

IDENTIFICATION OF CAMPYLOBACTER PYLORI......................................... Enzyme Profiling.................................................................................. Cellular Fatty Acid Analysis........... ..... ..................................... ............... Restriction Endonuclease DNA Analysis and Protein Electroph ore sis . ............. . .. CONCLUSION ........................................................................................

INTRODUCTION Scientists have observed spiral organisms in the gastric mucosa of animals and humans for over seventy-five years (25, 37, 98, 112, 113, 120), but not until the relatively recent reports by Warren & Marshall (133) was a relation ship between gastric disease and these bacteria demonstrated. The realization 249

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that gastritis and gastrointestinal ulcerative disease could be infectious prompted an unprecedented amount of active investigation over the past six years. As a result, a number of excellent current reviews deal with the association between gastric disease and the bacterium, Campylobaeter pylori (5, 7,26, 27, 41, 46, 54, 138). This review focuses on the bacteriology of the organism as well as methods of diagnosis and identification. In mid-1989, during the writing of this review, Campylobaeter pylori was appropriately transferred to a new genus,Helieobaeter (40). Because all the literature to date uses the older Campylobaeter nomenclature, Campylobaeter pylori rather than Helieobaeter pylori is used in this article. BACTERIOLOGY OF CAMPYLOBACTER PYLORI Morphological Characteristics C. pylori is a curved or S shaped gram-negative bacillus with 4-6 lophotrichous sheathed flagella. The bacteria are approximately 0.5 /-Lm wide and can vary from 2-3 /-Lm in length (45). Bacteral cells in older cultures and those found intracellularly tend to be coccoid forms rather than rods (8, 15). The cell wall is smooth and adheres closely to the cytoplasmic membrane, in contrast to Campylobaeter jejuni, which have a rough rugose cell wall (45). The most distinguishing structural characteristic of C. pylori is its sheathed flagella that terminate in a bulbous disc structure (39, 45). Structurally, the flagella of C. pylori consist of a filament composed of flagellin, a protein monomer (mol. wt. �51,000), a hook, a cell-anchored basal plate, and, unlike the majority of flagellated bacteria, a sheath (38). Morphologically, the flagellar sheath is continuous with the outer cell wall membrane; the flagellum is 30-35 nm in diameter; and the terminal bulb is 100 nm in diameter (38). Although the specific function or chemical composition of this structure has not been determined, it likely contains antigens that cross-react with anti serum against C. jejuni flagellin responsible for the lower specificity of serological tests that utilize whole cell preparations or extracts (28, 95). Metabolism and Physiology NUTRITIONAL REQUIREMENTS The specific nutritional growth require ments of C. pylori have not been determined, although several important factors of their cultivation have become evident. Early investigators observed that whole blood or heme-supplemented complex basal media enhanced the growth of C. pylori (13,52,77). Subsequently,Buck & Smith (14) demon strated that serum,charcoal,starch,and hemin supplementation of complex basal media would also permit growth,although they did not consider hemin an absolute growth requirement; this was confirmed by others (53). While some of these supplements can serve as nutritional substrates, their primary

HEllCOBACTER PYLORI

251


function is thought to be detoxification and protection of the organism. Hazell et al (53) have demonstrated the growth of C. pylori on a semi-defined basal medium supplemented with bovine serum albumin and catalase and suggested that these two components protected C. pylori from the toxic effects of unsaturated fatty acids through absorption and prevention of peroxidation of saturated fatty acids by H202 and iron. METABOLlSM C. pylori has been shown to produce a number of preformed enzymes that include short-chain fatty acid esterases, arylamidases, ami nopeptidases, alkaline phosphatase, catalase, oxidase, deoxyribonuclease, and most notably urease (81, 84, 86). It does not utilize carbohydrates either fermentatively or oxidatively. The specific metabolic mechanisms by which C. pylori obtains and utilizes carbon are not known, although the bacterium probably utilizes amino acids, TCA cycle intermediates, and lipids (53). The use of proton (' H) nuclear magnetic resonance (NMR) spectroscopy of brain heart infusion broth supplemented with 10% horse serum before and after growth of C. pylori has revealed that the bacterium metabolizes lactate and alanine (24). Major products of metabolism are acetate, succinate, glycine, and citrat�:. Similarly, multinuclear NMR in the same medium enriched with potential substrates showed that formate, a-ketoglutarate, and lysine were completely metabolized, but no products were detectable. TCA cycle activity was furthe:r evidenced through the addition of fumarate and malate to the base medium. Malate spurred production of fumarate and, conversely, malate production was observed when fumarate was added in excess. Interestingly, 13C-Iabelkd acetate was not metabolized by C. pylori, suggesting the lack of an acetatl� transport system or the absence of acetyl-CoA synthetase. Supplementation of the medium with serine increased production of succinate and ammonia detected by 'H NMR and 14N NMR, respectively. This finding suggests that in addition to its urease activity, C. pylori may have a general deaminasf: enzyme system and a serine-isocitrate lyase pathway. Addition of urea resulted in the production of ammonia. detected by 14NMR, and bicar bonate, detected by 13C NMR. In addition to bicarbonate, a resonance at 124.9 ppm was detected using 13C-Iabelled urea. This preliminary observa tion in conjunction with formate utilization suggests that C. pylori also possesses an active C-l metabolic pathway.

The most distinguishing metabolic characteristic of C. pylori is the production of a unique and very potent urease. The urease of C. pylori has been well characterized by Mobley et al (86). The average rate of hydrolysis of urea by cell lysates was 36 ± 26ILmol of NH3/minlmg protein, which is more than twice that of Proteus mirabilis. The urease of C. pylori is similar to other ureases in that it is a nickelUREASE AND NITROGEN METABOLISM


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requmng enzyme with a molecular weight of 625,000; although several investigators have demonstrated urease activity in proteins of lower molecular weight, which suggests a subunit structure (28, 76). The Km of the enzyme was found to be 0.8 ± 0.1 mM urea with an optimal temperature of 45°C and an isoelectric point of 5.9, a characteristic that separates it from the ureases produced by closely related organisms isolated from ferrets and baboons (124). The optimum pH reported by Mobley et al (86) is 8.2, although a second pH optimum in the acidic range, approximately pH 5, has been demonstrated by 14N NMR (24). Subunits of urease have been shown to be associated with both the outer-membrane membrane and flagella of C. pylori; one of these proteins, protein 2, is both unique and a major antigenic determinant for the organism (28). Despite the obvious importance of this enzyme in terms of anabolic energy expenditure, the specific function of urease remains speculative. Three poten tial functions of urease have been proposed and include (a) provision for nutritional nitrogen in the form of ammonia (35), (b) protection against gastric acid (41), and (c) involvement in the pathogenesis of gastric ulcer (51). Metabolically, C. pylori does possess an NADPH-dependent glutamate de hydrogenase system for utilization of ammonia in the synthesis of glutamate from a-ketoglutarate (35). Although plausible, the fastidious nutritional re quirements of C. pylori, specifically its requirement for complex culture media components, suggests that ammonia is not an essential or preferred nitrogen source. Protection against gastric acid damage through the produc tion of an alkaline ammonia cloud around the organism has been suggested as a primary function of the urease enzyme (41). While this postulate would explain survival in the acidic gastric environment, C. pylori is found in close association with the gastric epithelial cells and mucin layer that provide a relatively neutral environment (119). Similarly, the proposed role of urease in ulcer pathogenesis (51) should be viewed as a potential coincidental rather than primary function of the enzyme. Bacterial energy expenditure of the magnitude required to synthesize an enzyme-system of the complexity and distribution of C. pylori urease is usually only associated with essential structural or energy-producing cellular components. The location and dis tribution of C. pylori urease over the entire cell surface (9, 28) might permit the generation of an electrochemical gradient via NH4+ production across the cytoplasmic membrane, which could be utilized as an energy source. Such a mechanism has been proposed for the urease of Ureaplasma ureolyticum (111). ADAPTATION AND SPECIALIZATION One of the most interesting aspects of C. pylori is its ability to survive in the hostile gastric environment. This survival can be largely attributed to the development of several specialized

HELICOBACTER PYLORI

253

characteristics that have permitted a high level of adaptation common among organisms that inhabit gastrointestinal mucus

(68). These characteristics in

clude a spiral or helical morphology in conjunction with motility that facili tates movement through viscous environments such as mucus

(52, 68).

Another common feature or adaptive mechanism is microaerophilism. Ox ygen concentrations at the gastric epithelial surface and mucus layer are low

C. pylori. These characteristics in conjunction with a potent urease indicates an organism that is highly adapted for its environment, as evidenced by C. pylori'S apparent organotropism for gastric mucosa (8, 62, 138).


but not totally anaerobic, which is similar to the optimum for growth of

Potential Virulence Factors Many bacteria possess factors in addition to specialized adaptive mechanisms, which when produced under appropriate circumstances in a susceptible host, result in pathogenesis and disease. Several potential virulence factors have been identified for

C. pylori, although only a limited number of definitive

experiments with strains possessing and lacking a specific factor have been performed.

ADHERENCE FACTORS

Although adherence factors are not considered

pathogenic in and of themselves, they are discussed in this section because of their critical importance in initial colonization of host mucosa that is essential to subsequent pathogenesis.

C. pylori is found in close association with (52). Occasionally,

gastric mucosa cells, particularly at intracellular junctions

a cupping indentation or a thickening of the gastric epithelial cell at the point

(8, 46), suggestive of specific C. pylori adhesions or adher ence factors. Hemagglutinin activity has been demonstrated in C. pylori in a variety of erythrocyte species (92). Evans et al (32) have characterized a

of bacterial cell attachment has been observed

binding of gastric epithelial cell surfaces with

fibrillar hemagglutin that binds specifically to N-acetyl neuraminyl-Iactose and have proposed that this moiety is the primary adhesion of

C. pylori that (71)

mediates gastric mucosal colonization. Independently, Lingwood et al

have reported the isolation of a novel glycerolipid that they propose is the specific gastric epithelial receptor for

C. pylori adherence. Unfortunately, the

presence of an N-acetyl neuraminyl-Iactose moiety was not determined. Several in vitro models utilizing mammalian cell lines have been developed

C. pylori investigation (94). Using a Y-l mouse adrenal cell line, Evans et (31) have further substantiated the N-acetyl neuraminyl-Iactose-binding hemagglutinin as the primary adherence factor of C. pylori. In this model, C. pylori adherence was rapid, neuraminidase sensitive, and was successfully for al

blocked by a sialoglycoprotein rich in N-acetyl neuraminyl-lactose.

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CYTOTOXIN

Leunk et al (70) and others

(56) have demonstrated nonlethal

vacuolization and cytopathic effects in a variety of mammalian cell lines by

broth-culture filtrates of Campylobacter pylori. Approximately one-half of the isolates tested from different geographical regions produced the cytotoxic effect following concentration from culture filtrates. The cytotoxin was shown to be a heat-labile protein of molecular weight> 105 The activity was neutralizable by specific antisera to either culture filtrates. Figura et al (36) have shown a statistically significant difference in cytotoxin production by C. pylori isolates from patients with peptic ulcers versus patients with chronic gastritis. Similarly, using the gnotobiotic piglet animal model (63), Eaton et al (29) have shown a correlation between cytotoxin production and infection rate, although a much stronger correlation was found with motility.


.

C. pylori produces an extracellular protease that degrades pig gastric mucin (114, 118). This protease, when released by C. pylori in gastric mucosa, has been suggested to initiate the breakdown of mucin. Mucin, in tum, liquifies, looses its structure, and no longer serves as GASTRIC MUCIN PROTEASE

an effective barrier to the back-diffusion of hydrogen ions. The optimum activity of the enzyme was found at pH 7.0 and a temperature of 37°C with an

apparent Km of

0.71 gIL for gastric mucin.

UREASE As mentioned earlier, the potent urease of C. pylori has been proposed as a virulence factor (51). In essence, the hydrolysis of urea by C. pylori results in the production of ammonia that accumulates in the protective gastric mucin layer, causing a loss of ionic integrity (125). This ammonia induced change might explain the hypochlorhydria seen in C. pylori associated disease as well as permit the back-diffusion of hydrogen ion and

resulting gastric epithelial cell damage. This postulate does offer a plausible explanation for the development of gastritis and peptic ulcer disease, although some investigators have contended that ammonia is not toxic in the stomach (46). Cave & Vargas (16) demonstrated that whole or sonicated C. pylori cells can dramatically inhibit stimulated acid secretion from rabbit parietal cells. Using the uptake of 14 C-aminopyrine as an indirect assay, they found that the protein inhibitor was as effective as 10-4 molJI ACID SECRETION INHIBITOR

cimetidine for inhibiting acid secretion from parietal cells. They suggest that this protein inhibitor may be responsible for the hypochlorhydria seen during acute C. pylori infection. HEMOLYSIN Wetheral & Johnson (136) demonstrated a weak cell-free hemolysin in some strains of C. pylori. The hemolysin was active on human,

HEUCOBACTER PYLORI

255

horse, guinea pig, rabbit, and sheep erythrocytes and was not associated with ureolytic activity. DIAGNOSIS


Gastric Biopsy

Gastric biopsy, primarily of the antrum, has been the standard procedure for detecting C. pylori and a requirement, through histologic examination, in defining disease. Both the organism and polymorphonuclear leukocytes, indicative of gastritis, have been shown to be patchy and focal in their distribution in the gastric mucosa (3, 6, 18, 43, 60, 90, 123). This characteris tic can lead to sampling error resulting in incorrect diagnosis as well as confusion in the evaluation of new methods of diagnosis if histologic ex amination is used as the reference method. For these reasons, at least two biopsy specimens should be obtained at endoscopy with multiple sections being examined histologically for both the organism and the presence of gastritis (6, 90). The histologic staining of gastric biopsies is the standard and most frequently employed method for the detection of C. pylori. A variety of stains have been utilized for visualization of the organism on the gastric mucosa; the Warthin-Starry silver stain is the first and most frequently reported (133). However, the Warthin-Starry method is both time consuming and difficult and is not required to demonstrate the organism in gastric biopsy material. Some investigators (58, 123) have found hematoxylin and eosin, the standard histologic stain, to be equivalent to silver staining, although the organism is less discemable when compared to either Giemsa (104, 138) or acridine orange staining (132). Giemsa staining offers the advantage of preservation of morphology and permanence, while the acridine orange fluorescent method is rapid and easy to perform, on not only tissue sections, but on ground-culture specimens as well. Other nonspecific methods that have been successfully utilized for the detection of C. pylori include staining with carbol-fuchsin ( l 09), Gram (126), Gimenez (80), and phase contrast microscopy (102). Although the preceding methods all enhance visualization of C. pyLori, they are relatively nonspecific, staining any bacteria present in the gastric mucosa or introduced as a contaminant during the endoscopy procedure. Specific immunofluorescent methods have been described using either poly clonal antisera (115, 121) or monoclonal antibodies (30) to C. pylori. In addition, Negrini et al (93) have recently developed a rapid and highly specific indirect immunoperoxidase test using a monoclonal antibody on gastric brushings. As with all microscopic techniques, morphology as well as staining characteristics are critical for accurate determinations.

MICROSCOPIC EXAMINAnON


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CULTURE In conjunction with histologic staining, culture has been the reference method for detection and confirmation of C. pylori. Although essential for comprehensive diagnosis and evaluation, i. e. identification, characterization, and antibiotic susceptibility testing, culture of C. pylori has not proven to be the most sensitive method of detection. In addition to potential sampling error, successful culture of C. pylori from biopsy material can be confounded by prior antibiotic or bismuth compound-use by the patient, use of antiseptics or other bacteriostatic agents during endoscopy, inappropriate handling and transport of the specimen, as well as inadequate laboratory processing. Despite these problems, high recovery (> 90%) rates have been reported by a number of authors (43, 58, 102, 116, 123) when compared to histological staining. A combination of culture with histologic staining, however, provides the best sensitivity for both diagnosis and new method evaluation (43, 76, 116). Ideally, a biopsy specimen should be processed as soon as possible in order to optimize the chances for recovery of C. pylori, but due to the frequent nonadjacency of endoscopy suites and microbiology laboratories, transport of the specimen is required. Several transport media have been utilized, and they include nutrient broth (55), brucella broth (123), brain heart infusion (97), trypticase soy broth with 10% horse serum and 5 mM urea (19), 20% glucose (43), and physiological saline (19, 61), although C. pylori has been shown to lose viability in the latter (49). In addition, a selective biphasic transport medium has been suggested that can be used as an isolation medium following plating of the specimen (135). Once obtained, specimens can be held or transported at room temperature or 4°C for approximately 4-5 hours (19, 43); however, rapid processing « 2 h) from transport medium will decrease overgrowth by contaminants and increase C. pylori viability. Initial process ing should involve rubbing of the biopsy directly on to solid culture media, and mincing, or preferably grinding, which reportedly results in heavier, more uniform growth (43). A variety of primary culture media have been utilized for the growth of C. pylori and include enriched, selective, and differential agars and broths. While this reflects a lack of knowledge of the specific nutritional requirements of C. pylori, several basic principles have become apparent. The basal media, out of necessity, should be complex with brucella agarlbroth (88, 117, 123)-brain heart infusion (103), Mueller-Hinton (14), and trypticase soy (19) have been used successfully. All complex basal media require supplementation, preferably with fresh whole blood (horse, sheep, rabbit) at concentrations ranging from 7-20% (19). Although a blood agar medium is used extensively with isolated colonies demonstrating characteristic colonial morphology and weak hemolysis, chocolate agar is also used widely. Other supplements to the above basal media that support the growth of C. pylori,


HEUCOBACTER PYLORI

257

although usually not as well, include serum, starch, charcoal, bovine serum albumin, and catalase (14, 53). In addition to one or more of the above primary enrichment media, a selective medium should be used for suppres sion of re:;piratory flora and other potential contaminants. Selective supple ments include vancomycin, for inhibition of gram-positive bacteria, and a combination of trimethoprim with polymyxin B or nalidixic acid, or cefsulo din alone for inhibition of gram-negative bacteria. Either cycloheximide, nystatin, or amphotericin B are used for inhibition of fungi (43, 65). Queiroz et al (103) have used a differential and selective medium. Care should be taken in selecting the concentration and type of antibiotic used to avoid C. pylori inhibition. Although not yet tested on clinical specimens, broth (88, 97) and biphasic systems (117) have been suggested as an alternative to solid agar media for the isolation of C. pylori from gastric biopsy specimens. Regardless of the specific culture system, fresh « 2 weeks old) media are essential for successful recovery. Culture media, once inoculated, are incubated under moist microaerophilic conditions (7-10% CO2, 5% 02) and, in the case of liquid broth systems, with continuous shaking to improve gas dispersal (88). The microaerophilic requirement can be achieved through the use of commercial gas-generating systems, evacuation of gas jars and replacement with an appropriate gas mixture, or the use of a humid carbon dioxide incubator. Isolates of C. pylori will not remain viable if left under aerobic conditions for extended periods. The optimum growth temperature for C. pylori is 37°C, although it will grow at 42°C. It does not grow at room temperature. DIRECT UREASE TEST The early observation that C. pylori produced a potent ure:ase (96) led McNulty & Wise (83) to utilize this characteristic to detect the organism directly in gastric biopsy tissue. This initial success as well as the ease and rapidity of this test has led to its widespread use and modification by a number of investigators, including its originator (82). All of the direct urease tests rely on the existence of preformed urease in the presence of C. pylori in gastric biopsy specimens, precluding a requirement for growth of the bacterium. In the presence of exogenous urea, if the tissue is placed in a urea-containing broth or agar, hyrolysis occurs with formation of ammonium ions, alkalinization, and an increase in pH, which can be visually detected th.rough the use of pH indicator, usually phenol red, that results in a color change in the medium from beige to pink/purple. Variations in test methods have primarily included changes in medium composition or tempera ture and length of incubation prior to reading. In general, the sensitivity of the direct urease tests increases as the length of time prior to reading the color change increases; the specificity concomitantly decreases. The increased time required for urea hydrolysis allows the pH indicator to change in specimens

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Table 1

Detection of Campylobacter pylori by the gastric biopsy urease test

Author Das et al

Method

(21)

Sensitivity

Specificity

tested

(%)

(%)

Timing

Christensen's

50

74

88

24h

Christensen's

597

92

99.4

71% 2h

Modified

847

96

100

74% 2h

CLO test

141

98

100

75% 20 min

Hazell et al (53)

Microtiter

346

91

100

75% Ih

Morris et al (91)

CLO test

70

96

100

4h

Vaira et al (127)

Rapid urease

256

89

100

100% in 4h

Arvind et al (4)

1 minute test

40

90

100

100% in 1m

Westblom et al (134)

Urea broth

75

67

100

83

74

McNulty et al (82) Marshall & Langton (74)


Number

49

62

100

93

100

CLO test

101

91

91

24h

Microtiter

151

92

86

24h

Urea Schnell & Schubert (116)

25°C 55°C

49

Urea

Abdalla et al (I)

Ih 24h

-

-

Ih Ih

with low numbers of C. pylori, but false positive tests could result from urea hydrolysis by low numbers of contaminating urease-producing bacteria or autohydrolysis. As shown in Table 1, all of the direct urease tests demonstrate good sensitivity and specificity when compared to microscopic and/or culture methods. In addition, the majority of positive tests occur within 1-2 hours of inoculation, yielding a rapid diagnosis at the time of endoscopy. Variations in speed, sensitivity, and specificity between tests have been attributed to differ ences in media composition, and temperature of incubation. Christensen's urea medium includes a nutrient base potentially permitting growth of con taminants and false positive reactions (82), while others utilize only un buffered urea and a phenol red indicator (1, 12, 79). These also have the potential for false-positive reactions with urease from organisms other than C. pylori. Sodium azide has been used as a selective agent (50). Several authors have suggest ed a buffered 2% urea with indicator as the optimal medium for performance of the test (39, 82). The optimum temperature of C. pylori urease is 45°C, and methods that incubate cultures at temperatures closer or at this optimum have shown increased ·sensitivity and specificity when compared to tests incubated at room temperature (1, 82). DETERMINATION

OF

GASTRIC

UREA

Marshall & Langton (74) have

demonstrated the potential utility of the determination of urea concentration in the gastric secretions of fasting patients for the detection of C. pylori. As the authors suggest, this method offers the potential for diagnosis of C. pylori without biopsy in those patients in whom it is contraindicated (i.e. thrombo-

HEUCOBACTER PYLORI

259


cytopenia, bleeding disorders) and patients who have a nasogastric tube in place or are vomiting. Using a value of < Immll of urea as the differential concentration, Marshall & Langton obtained a specificity of 100% and a sensitivity of 90%. In an analysis of gastric aspirates from 99 endoscopy patients, using a urea (mg/dl)/chloride (mEg) ratio in an attempt to account for potential gastric juice dilution prior to or during endoscopy, this laboratory (J. Dick, unpublished data) found a sensitivity and specificity of 75% and 86%, respectively, using a ratio of 5 as the cutoff. Serology C. pylori elicits both a local and systemic response in hosts that includes an elevation nn specific antibody of the serum IgG and IgA classes as well as secretory IgA and low levels of gastric IgM (105). This observation has resulted in the development of a variety of serological tests for the organism. Serological tests offer advantages over the biopsy tests in that they are noninvasive, relatively simple, rapid, and cost-effective. Serological tests in addition to diagnosis are particularly useful in screening large numbers of individual:, for epidemiological studies (85, 107), and potentially for monitor ing therapy (128). Several techniques have been utilized for determining C. pylori serology that include complement fixation (58, 131), hemagglutination (75), immunoblot (59, 130, 131), time-resolved fluoroimmunoassay (2), and, as shown in Table 2, the most widely utilized, enzyme-linked immunosorbent assay (EUSA). Although determination of serum IgA titers has been shown to correlat,e well with the presence of C. pylori (11, 105, 130), serum IgG is the preferred antibody class for serological determinations (10). Table 2. shows the sensitivity, specificity, and predictive values for a number of published ELISA IgG studies. In all instances, results were compared to biopsy determinations, e.g. microscopy and/or culture, or the l3e-urea breath test. The variation in sensitivity, specificity, and predictive values of the different tests can be attributed largely to the use of different antigen preparations as well as the method used for reference comparisons. Many assays utilize whole cells (one or more strains) (57, 105), sonicates (11, 99), or acid-glycine extracts (42). Unfortunately, C. pylori shares some antigenic determinants with other bacteria, most notably C. jejuni (69, 95), which has resulted in cross reactivity in many of these systems. In an effort to avoid this problem, some investigators have chosen more than one threshold ELISA value based on increasing either the specificity and positive predictive value (increase the cut-off threshold) or the sensitivity and negative predictive value (decreasing the cut-off threshold), depending on the particular applica tion of the test. Alternatively, a number of investigators have utilized purified or partially purified antigen preparations that have been shown to improve the diagnostic value of this method (20, 22, 33). The majority of these newer

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Table 2

Detection of Campy lobaeter py lori by ELISA JgG serology


Author

Antigen

Number of

Sensitivity

Specificity

Positive

patients

(%)

(%)

(%)

(�

74 100

69 100

101

97 86

98 80

99 85

97 78

98 91

100

100

71

1 00 1 00 82 82

73 79 92 94

89

1 01 1 01

93 93 93 1 00

7' 7'

Booth et al (11 )

Whole cell

Iones et al (58)

Whole cell

Rathbone et al (105)

Whole cell

Paull et al (99)

Whole cell

Goodwin et al (44)

Acid glycine extract

LoffeId et al (72)

Whole cell

Dent et al (22)

Urease

202

Bolton et al (l0)

Surface antigen

1 99 1 99

Acid glycine extract Crude urease Evans et al (34)

High molecular weight

96 64 73 48 160 70

199 300

100 97 93 84 81

97 98.7

90 1 00

cell-associated protein

antigen preparations include the urease or subunits of the urease enzyme that are unique to C. pylori (8, 28). Care should be taken in the purification and selection of specific C. pylori proteins for use in serological tests in assuring that a specific antigen is not only unique for C. pylori, but also is universally found in all strains of the species (34). Urea Breath Test

The potent urease activity of C. pylori has permitted the development of noninvasive tests based on the rapid hydrolysis of urea to carbon dioxide and ammonia when it is placed in contact with gastric mucosa colonized with the organism. Unlike the direct biopsy urease test, the breath tests are noninva sive, relying on the production of isotopic, either l3C or 14C, carbon dioxide from labelled urea. Isotopically labelled urea is given to the patient, usually following a meal to delay gastric emptying, and the appearance of labelled CO2 in the breath is monitored with time using either gas-isotope ratio mass spectrometry in the case of I3C-urea or a liquid scintillation counter for 14C-urea after trapping in an alkaline solution (78, 106). A clear delineation in terms of labelled CO2 production separates individuals infected and those not infected with C. pylori, and a semiquantitative relationship between the number of organisms present and the concentration of labelled CO2 generated during the 30- to 60-minute time course has been shown with the 14C-urea test (106). Both tests measure active infection or colonization and are very useful in monitoring response to therapy as well as diagnosis. The 13C-urea breath test described initially by Graham et al (47, 48) offers the advantage of a

Neg;

9' 8' 5' 9:

91 9l

HEUCOBACTER PYLORI

261

stable nonradioactive isotope that can be used in children and pregnant women but unfortunately is costly in terms of the isotope and the equipment required fer detection. Alternatively, the 14C-urea breath test is less expensive and more sensitive but does involve, albeit low, exposure to a long-lived radioisotope.


Nucleic A.cid Hybridization and Antigen Detection Methods

The majority of researchers in this field are actively investigating the utiliza tion of nucleic acid hybridization and monoclonal antibody antigen detection systems, but unfortunately no large clinical studies using an antigen detection system or probe has appeared in the literature. For this reason, the comparison of nucleic acid probes or antigen detection methods for C. pylori in terms of 2 sensitivity and specificity is premature. A number of 3 P-Iabelled and nonra dioactive genomic DNA probes have been developed and evaluated in both dot-blot (137) and in situ hybridization systems (129). Alternatively, Moroto mi et al (89) have developed a C. pylori-specific oligonucleotide probe to 16S ribosomal RNA based on detailed sequence data (110), which is rapid, potentially simpler, and more sensitive than DNA dot-blot assays. Similarly, the use of oligonucleotide primers and DNA probes in the polymerase chain reaction system offers the potential of maximum sensitivity and specificity in detecting C. pylori, including nonculturable forms that might exist in the oral cavity, stool, or environment. Species and strain-specific antigens have been successfully cloned into E. coli K-12 (17). In addition to utilization as DNA probes, cloned species-specific surface immunogenic proteins and monoclo nal antisera have potential for use in antigen-detection systems as well as improved serological assays (28, 101). IDENTIFICATION OF CAMPYLOBACTER PYLORI

Since the initial description of the bacterium, evidence has mounted that it is not related to other members of the genus Campylobaeter. Conventional biochemical characteristics, protein profiles, cellular fatty acid and quinone composition, ultra structure, and nucleic acid analysis have all suggested that it is taxonomically distinct from previously described bacteria. As a result, C. pylori has been placed in a new genus Helicobacter along with a related gastric bacterium found in ferrets (40). A number of identification methods have been developed for both species and strain identification of C. pylori following isolation. All of the previously described methods such as monoclonal fluorescent antibody and nucleic acid hybridization assays are applicable for identification, but this section deals with methods that have been utilized on cultured bacteria. C. pylori can be readily ide:ntified on the basis of characteristic colonial and microscopic

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morphology, a positive oxidase and catalase test, and the rapid hydrolysis of urea. All of these tests are currently available in routine clinical microbiology laboratories.


Enzyme Profiling

A number of investigators have utilized commercially available enzyme test kits that detect the presence of preformed enzymes through the use of chromogenic substrates, for the identification of C. pylori. Megraud et al (84) tested 78 enzyme substrates, not all of which were commercially available, found 31 enzymes present in at least one of the twenty isolates tested. Enzymes that were consistently present in all strains tested included arylarni dases of N-benzoyl-Ieucine, S-benzyl-cysteine and L-phenylalanine-L proline, esterases of short chain (C-4 to C-lO) fatty acids, transpeptidase, alkaline and acid phosphatase, and phosphoamidase. Similarly, McNulty & Dent (81), using a different commercial system, found consistent production of r-glutamyl aminopeptidase, leucine aminopeptidase, and alkaline phospha tase as well as urease, catalase, oxidase, and deoxyribonuclease using con ventional methods. The use of different commercially available systems have yielded similar results, although enzyme activity can vary from one com mercial kit to another depending on the particular substrate and reaction conditions (Sl, 122). On the basis of variations in esterase (C-4 and C-S), naphthol-AS-B1-phosphohydrolase, and arylamidase activity, two biotyping schemes have been suggested for strain differentiation (64, 108). Cellular Fatty Acid Analysis

Cellular fatty acid analysis through the use of gas-liquid chromatography is an effective method for identification of C. pylori (45, 66; J. Dick, unpublished information). Using this technique, investigators can readily differentiate C. pylori from members of the genus Campylobacter as well as other closely related bacteria on the qualitative presence of methylene octadecanoic acid (a 19: 0) and quantitative differences in tetradecanoic acid (14: 0), hexadecanoic acid (16 : 0), octadecenoic acid (18 : I), and octadecanoic acid (18 : 0), and the absence of 3-hydroxy-tetradecenoic acid (3-0H-14: 0) and hexadecanoic acid (16: 1). The most unique cellular fatty acid of C. pylori is 3-hydroxy octadecanoic acid (3-0H-18: 0). This particular fatty acid is present in signifi cant amounts in only Brucella, Francisella, and Acetobacter. Lambert et al (66) and subsequently Goodwin et al (44) have proposed the classification or grouping of campylohacters and related bacteria into 11 (A-K) groups on the basis of cellular fatty acid composition. While providing excellent species differentiation, cellular fatty acid analysis is not useful for strain differentia tion.

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Restriction Endonuclease DNA Analysis and Protein Electrophoresis

Analysis of C. pylori proteins using polyacrylamide gel electrophoresis is useful in species identification (84, 100). Strains of C. pylori yield seven common major-protein bands but are not significantly diverse to permit strain differentiation. In contrast, Langenberg et al (67), using restriction endonu clease DNA analysis, found significant strain differences in HindIII digests and suggested this method for epidemiological studies. Majewski & Goodwin (73) found similar variation in HindIII digest of DNA from C. pylori isolates, but found variation with time in consecutive isolates from the same patient following treatment and suggested that restriction endonuclease DNA analysis may not be a suitable method for epidemiological studies. CONCLUSION

The realization that gastritis and peptic ulceration could have infectious disease etiology has resulted in an entirely new area of investigation for microbiology. Clearly, significant strides have been made in the development of method:; of detection and identification of the causative agent. Similarly, a better undl!rstanding of the bacteriology of C. pylori has also resulted. As a pathogen, C. pylori has much to teach us and we have much to learn concerning basic physiology, metabolism, and pathogenicity. ACKNOWLEDGMENTS

I appreciate the cooperation and support provided by Drs. Ray R. Arthur, John G. Bartlett, Michael Gamcsik, Harry L. T. Mobley, Gerson Paull, William J. Ravich, James W. Rawles, and Michael J. Zinner in many of the investigations. I am particularly grateful to Drs. John H. Yardley and Leslie L. Walters for initially spurring my interest in this organism. I thank Alan O'Neill for his superb secretarial support. Literature Cited I. Abdalla, S . , Marco, F., Perez, R. M., Pique, J. M., Bordas, J. M., et al. 1989. Rapid detection of Campylobaeter pylori colonization by a simple biochemical test. J. Clin. Microbiol. 27:2604-5 2. Aceti, A., Pennica, A., Leri, 0., Cafer ro, M.,. Grilli, A. , et al. 1 989. Time resolved fluoroimmunoassay for Campy/obacter pylori antibodies. Lan cet 2(8661):505 3. Anderson, L. , Holek, S . , Poulsen, C., Elsborg, L., Justen, T. 1 987. Campylabaeter pyiaridis in peptic ulcer

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Topographic association between active


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