Digestive Diseases and Sciences, Vol. 36, No, 8 (August 1991), pp. 1084-1088
Seroepidemiology of Helicobacter pylori Infection in India Comparison of Developing and Developed Countries DAVID Y. GRAHAM, ERVIN ADAM, GURUNATH T. REDDY, JAI PRAKASH AGARWAL, ROHIT AGARWAL, DOYLE J. EVANS, JR., HODA M. MALATY, and DOLORES G. EVANS
Helicobacter pylori (previously Campylobacter pylori) is now accepted as the major cause of type B gastritis and thus what is known about the epidemiology of type B gastritis can reasonably be transferred to H. pylori. We used a specific ELISA for anti-H, pylori lgG to study the prevalence ofH. pylori infection in a population of lower socioeconomic class from Hyderabad, India. The results from India were compared to studies from other parts of the world. Two hundred thirty-eight individuals ages 3 to 70 participated. The frequency ofH. pylori infection increased with age (P < 0.01) and was >80% by age 20. H. pylori infection was present in 79% of the population studied; there was no gender-related difference in prevalence of H. pylori infection. IgG antibo~' against hepatitis A (HA V) was rapidly acquired in Hyderabad; in a subset of 58 children between the ages o f 3 and 21 tested, the frequency of anti-HA V was 98.2%. The prevalence of H. pylori infection increases with age in both developed and developing countries. The high age-specific prevalence of H. pylori infection in developing countries is probably a reflection of the lower socioeconomic level of those areas. KEY WORDS: Helicobacter pylori; seroepidemiology; race; age-specific; socioeconomic group; hepatitis A.
Helicobacter pylori (previously Campylobacter pylori) is now accepted as the major cause of type B gastritis and thus what is known about the epidemiology of type B gastritis can reasonably be transferred to H. pylori. For example, we know that gastritis is more frequent in those of low socioeconomic class, large families, and crowding, and varManuscript received July 11, 1990; revised manuscript received January 22, 1991; accepted January 28, 1991. From the Department of Medicine and Division of Molecular Virology, Veterans Affairs Medical Center and Baylor College of Medicine, Houston, Texas; and Deccan Pathological Institute, Osmania General Hospital, and the Niloufer Hospital, Hyderabad, India. This work was supported by the research funds from the Department of Veterans Affairs; by grant DK 39919 from the National Institute of Diabetes and Digestive and Kidney Diseases, and by the generous support of Hilda Schwartz. Address for reprint requests: Dr. David Y. Graham, V. A. Medical Center (111D), 2002 Holcombe Blvd., Houston, Texas 77030.
1084
ies depending on the ethnic population studied. Ethanol, nicotine, and nonsteroidal antiinflammatory drug use do not play an important role (1-4). The incidence of gastritis varies both between and within countries (5). H. pylori infections are worldwide. Most reports have emphasized H. pylori infections in patients presenting for endoscopy and thus provide little information about the frequency in the general population. Others have used sera obtained from blood donors, which are often very select groups. We used a sensitive and specific ELISA to detect anti-H, pylori IgG antibodies against purified highmolecular-weight cell-associated antigens (HMCAP) to evaluate the prevalence of H. pylori infection (6). We also compared the rate of acquisition of H. pylori antibody with that of antibody to hepatitis A. We report the prevalence of H. pylori infection Digestive Diseases and Sciences, Vol. 36, No. 8 (August 1991)
0163-2I16/91/0800-1084506,50/09 1991PlenumPublishingCorporation
SEROEPIDEMIOLOGY OF Helicobacter pylori INFECTION
in a population of lower socioeconomic class from Hyderabad, India, and compare these results to studies f r o m o t h e r parts of the world.
M A T E R I A L S AND M E T H O D S Hyderabad is a cosmopolitan city in southern India with a population of approximately 4 million. Adults. The plan was to obtain serum specimens from approximately 20 individuals per age group. The study populations were obtained in July and August, 1988, from a walk-in outpatient medical clinic of the Osmania General Hospital, Hyderabad, India. Patients with any serious illness such as chronic heart, liver, renal, or lung disease were excluded. Patients were selected who (1) presented for minor illnesses or for follow-up during two days of the week (Tuesday and Thursday), (2) were within the age range(s) of the study, and (3) agreed to participate. Each patient was questioned concerning drug use, smoking, and symptoms referable to the upper gastrointestinal tract. Children. The study populations were obtained in February and March, 1989, from an outpatient pediatric clinic of Niloufer Hospital of the Institute of Pediatrics, Hyderabad, India. Children were selected who were within the age range(s) of the study, and parental consent was obtained. Both hospitals are public hospitals providing charity care to the lower socioeconomic class. Each patient had a blood sample obtained; the serum was separated and frozen until transfer to Houston, Texas. ELISA for Anti-H. pylori IgG. Specific details are published elsewhere (6); briefly, high-molecular-weight cell-associated protein obtained from n-octyl-glucoside extract of H. pylori cells was placed on an Agarose A-5m column and eluted with 0.05 M Tris C1 buffer pH 8.0. The two or three fractions possessing maximum urease activity were pooled. These fractions contain proteins in the molecular weight range of 400,000 to 700,000. The final antigen preparation contained at least two proteins, and analysis of ELISA-positive sera has demonstrated antibody against both of these major proteins. The ELISA was done in 96-well Microtiter plates (Linbro Scientific Co., Hamden, Connecticut) coated with HM-CAP diluted with PBS to approximately 7 ixg/ml. Sera diluted 1 to 50 and 1 to 100 were added and alkaline phosphatase-conjugated anti-human IgG (Southern Biotechnology, Birmingham, Alabama) was used as the detector. Each plate included HM-CAP ELISApositive and -negative control sera. The specificity and positive predictive value of the HM-CAP ELISA of the test were each shown to be 100%; the sensitivity is 98.7% and the negative predictive value 98.6%. There is no cross-reactivity with C. jejuni (unpublished studies). Antibody to hepatitis A (IgG) was determined on a random sample of 58 enrollees age 2-20 years. Antihepatitis A IgG was measured using the Abbott HAVABEIA assay. Data Analyses. The data was analyzed by logistic regression and Mantel-Haenszel chi square (SAS Institute, Digestive
Diseases
and
Sciences,
Vol. 36, No.
8 (August
1991)
TABLE 1. DISTRIBUTION OF THE STUDY POPULATION BY AGE AND SEX
Age range
Number
Male
Female
3-10 11-15 16-20 21-30 31-40 41-50 51-60 61-70
30 18 21 59 50 33 17 10
14 12 14 38 32 21 14 6
16 6 7 21 18 12 3 4
Cary, North Carolina). Age was a control variable in all analyses done. RESULTS
Two hundred thirty-eight individuals participated (Table 1). The male-female ratio was 1.7 to I. The frequency of H. pylori infection increased with age (P < 0.01) and was >80% by age 20 (Figure 1). There was no difference in prevalence of H. pylori infection between males and females (79% for both). Thirty-eight enrollees whose age was >20 had symptoms referable to the upper gastrointestinal tract; these symptoms were not the reason for visiting the outpatient clinic. The frequency of H. pylori infection in those with symptoms (84%) was the same as those without symptoms referable to the upper gastrointestinal tract (84%). There also was no difference in the frequency of H. pylori infection relating to whether the enrollee was a smoker or not. IgG antibody against hepatitis A (HAV) was rapidly acquired in Hyderabad. In 58 individuals 100 C.9
-r__ o Q..
80
-~ 6O
ON i
I
9
9
n =
238
~ 40
~ 2o n
o
...,
0
.....
10
, .....
20
, .....
,
.....
30 40 Age
,
. . . .
50
,
. . . . .
60
,
70
Fig 1. The age-specific acquisition of antibody to H. pylori infection is shown. The age ranges plotted are 3-10, 11-15, 16-20, 21-30, 31-40, 41-50, 51-60, and 61-70. 1 085
GRAHAM ET AL TABLE 2. SUMMARYOF SEROEPIDEMIOLOGICSTUDIES OF H. pylori INFECTION IN DEVELOPING AND DEVELOPED POPULATIONS Percent with antibodies to H. pylori (N in group) Age group Country
Australia Ref 18 Ref 14 Ref 7, 8 New Guinea Ref 14 Ref 14 New Zealand Ref 16 France Ref 15 England Ref 9 El Salvador Ref 18 Ethiopia Ref 18 Vietnam Ref 18
Population studied
10-19
20-29
30-39
40-49
50-59
Il (19) 5 (58) 0 (106) 59 (22) 20 33 (165) 16 (98) 4 (238) 43 (14)
0 (32) 18 (167) 0 (80) 66 (44) 38 45 (93) 25 (105) 19 (64) 25 (16) 47 (17) 12 (49) 75 (92) 75 (78) 85 (83) 75 (100) 81 (59)
13 (24) 14 (253) 0 (43) 58 (26) 28 54 (35) 27 (229) 19 (53) 47 (17) 41 (46) 22 (51) 78 (72) 83 (40) 88 (49) 73 (85) 92 (50)
17 (36) 27 (188) 0 (27) 75 (12) 54 33 (12) 33 (285) 16 (37) 50 (16) 24 (21) 17 (47) 85 (50) 75 (22) 95 (27) 75 (64) 88 (33)
30 (33) 37 (94) 11 (18) 64 (11) 40 0 (3) 35 (324) 40 (28) 33 (12)
White blood donors Unspecified, white?
4 (25)
Aborigines Papua, unadsorbed Papua, adsorbed Unspecified, blood donors? Bordeaux, outpatients Mixed
36 (112) 32 (175) 4 (113) 5 (22)
Immigrants to Australia Immigrants to Australia Immigrants to Australia Vietnam, blood donors
Ref 15 Ivory Coast Ref 15 Algeria Ref 15 Saudia Arabia Ref 11 India This study
0-9
Not specified Blood donors, outpatients Community-based healthy subjects Outpatients
13 (61) 55 (116) 45 (42) 42 (65) 60 (30)
45 (43) 75 (I00) 75 (59) 45 (! 37) 69 (39)
between the ages of 3 and 21, the frequency of anti-HAV was 98.2%. DISCUSSION
Published studies on the epidemiology of H.
pylori infection show considerable differences in the prevalence of antibodies to H. pylori in different populations studied (2, 7-18). The interpretation of these findings and the possible association of the infection with later sequelae of H. pylori exposure has its limitations due to two major factors: the representation of the population group studied, and the laboratory methods used for detection of specific antibodies to H. pylori (19). Nevertheless, a certain common pattern allows us to draw necessary further working hypotheses. The specimen collections that resulted in the data shown in Tables 2 and 3 include samples from different sources such as blood donors, newcomers to a given country, persons visiting outpatient health clinics for reasons other than gastrointestinal 1086
22 (18) 52 (21) 70 (7) 88 (17) 85 (53) 100 (17)
>60
20 (10)
33 (45) 47 (165)
24 (25) 55 (26) 80 (I1) 80 (45) 80 (10)
Totals
15 (154) 20 (785) 0.7 (277) 56 (157) 36 (483) 25 (1199) 21 (607) 40 (75) 43 (74) 18 (190) 53 (365) 73 (374) 79 (277) 72 (490) 81 (238)
symptoms, and from people presenting for regular periodic check-ups. It is not clear whom these groups represent, and socioeconomic differences and different exposures to infection are highly probable. Moreover, these serum collections include TABLE 3. PREVALENCEOF ANTIBODY TO H. pylori IN DIFFERENT POPULATION GROUPS IN NEW ZEALAND* Groups studied
Ethnic groups Maori Cook Island Samoan Tongan Occupational groups South Island meat inspectors Freezing works, veterinary surgeons Auckland meat workers Pig farmers Dairy farmers Pest control officers
Studied (N)
Infected (%)
192 85 129 56
21 39 44 70
18
17
18 80 68 20 14
44 41 13 20 21
*From reference 16. Digestive Diseases and Sciences. Vol. 36, No. 8 (August 1991)
SEROEPIDEMIOLOGY OF Helicobacter pylori INFECTION different ethnic groups with a prevalence ranging from 21% to 70%, such as in New Zealand (Table 3). Outpatient clinics in some countries may represent a mostly indigent population. Health-conscious persons visiting well-established health centers and high socioeconomic groups are probably not represented in any geographic area shown in Table 2. One problem in interpretation of data relates to the assays used to identify an H. pylori infection. The first-generation serologic tests employing whole bacteria, bacterial sonicates, or extracts of whole bacteria have all suffered from a high frequency of false positive tests caused by the presence of antibodies directed against antigen of C. jejuni or other bacteria containing antigens that cross-react with H. pylori (14, 19). The presence of cross-reacting antibodies appears to be particularly severe in studies using sera obtained from individuals living in developing countries. Recently, the suggestion was made that serologic tests should be restandardized for each population (14). Nevertheless, there are common patterns in t h e studies shown in Table 2. Evidently, the acquisition of H. pylori infection starts early in life and continues for several decades. Although the prevalence rates may be substantially different because of selection biases or methodological faults, the age-dependent trend is preserved. The difference in the prevalence ofH. pylori antibodies between groups also remains when samples are tested in the same laboratory. For example, the blood donors in Australia differ substantially from a group of Aborigines who were virtually not exposed to H. pylori infection and from ulcer patients who have a significantly higher rate of H. pylori antibodies--independent of age. Another example of sera tested in the same laboratory comes from France where blood donors in Vietnam or Algeria and the Ivory Coast had a substantially higher prevalence of antibodies to H. pylori than the local French population (15). Finally, Vietnamese who immigrated to Australia had H. pylori antibody rates significantly lower than blood donors in Vietnam. It is open to speculation if this difference is related to the assay per se or if there is a substantial socioeconomic difference between the local Vietnamese population represented by blood donors and emigrants. Despite the complexity of comparison of data from different geographic areas, the data support the concept that in some populations one major pathway of transmission is fecal-oral. The data also support the notion that the age-specific prevalence Digestive Diseases and Sciences, Vol. 36, No. 8 (August 1991)
of H. pylori infection, expressed as IgG antibody to H. pylori or those obtained using the [13C] urea breath test in Peru (13), in developing countries is related epidemiologically to the generally socioeconomic level of the geographic area. For example, Australian Aborigines showed two patterns: southern Aborigines had a higher rate of acquisition of H. pylori antibody than white Australians, as would be expected in a population of low socioeconomic status. In contrast, northern Aborigines appear to be a closed community in which H. pylori has not been introduced. The emerging pattern of the epidemiology of H. pylori infections is very reminiscent of the epidemiology of two other fecal-oral transmitted diseases, poliomyelitis and hepatitis A. For example, until the last century, poliomyelitis had always been a mild or subclinical disease in infants and as immunity is lifelong, older children and adults were immune. Improvements in the public environment, personal hygiene, sanitation, housing, and education resulted in an increasingly larger portion of the population that escaped exposure during infancy (20-22). This resulted in an even larger population of susceptible individuals in whom the disease would manifest as a paralytic poliomyelitis. The change to epidemics of paralytic poliomyelitis prompted a number of excellent epidemiologic studies proving an inverse relationship between the rate of acquisition of anti-polio antibody and standards of hygiene. In populations with poor standards, the steepest rise in antibody prevalence occurred in the youngest age group, whereas those with higher standards (eg, directly related to socioeconomic levels) experienced a delay with the steepest point of the curve in children of school age or adolescence. The differences in the prevalence of paralytic poliomyelitis were not based on geography per se but were related to standards and practices of hygiene and socioeconomic conditions. The epidemiology of hepatitis A was found to mirror that of poliomyelitis (23-26). It appears that the same factors that affected the age-specific prevalence of antibody to poliomyelitis (before vaccination) and to HAV also predict the age-specific prevalence of antibody to H. pylori. HAV is much more contagious than H. pylori and thus the rate of acquisition is more rapid and serves as a marker for poor sanitary conditions (11, 27). We postulate that, if both HAV and H. pylori were transmitted by the fecal-oral route, a similar shift in the age of acquisition of H. pylori infection could also be demon-
1087
GRAHAM ET AL s t r a t e d in the s a m e p o p u l a t i o n . C o n s e q u e n t l y , a shift in the o n s e t o f H. pylori i n f e c t i o n c o u l d d e l a y the o n s e t o f d u o d e n a l ulcer d i s e a s e a n d result in a r e d u c t i o n in the f r e q u e n c y o f gastric c a r c i n o m a b y d e l a y i n g the o n s e t o f the p r e c u r s o r lesion, a t r o p h i c gastritis.
ACKNOWLEDGMENTS The authors thank F. B. Hollinger for performing the hepatitis A antibody determinations.
REFERENCES 1. Graham DY, Adam E, Klein PD, Evans DJ Jr, Evans DG, Hazell SL, Alpert LC, Michaletz PA, Yoshimura HH: Epidemiology of Carnpylobacter pylori. Gastroenterol Clin Biol 13:84B-88B, 1989 2. Graham DY, Klein PD, Opekun AR, Boutton TW, et al: Epidemiology of Campylobacter pylori infection: Ethnic considerations. Scand J Gastroenterol 23 (suppl 142): 9-13, 1988 3. Massarrat S, Paidlik A, Pittner P, Schmitz-Moorman P, Wurbs M: The role of certain habits and various diseases in the occurrence of gastritis. Hepato-Gastroenterol 30:249, 1983 4. Edwards FC, Goghill NF: Aetiological factors in chronic atrophic gastritis. Br Med J 2:1409-1415, 1966 5. Cotran RS, Kumar V, Robbins SL (eds): Robbins Pathologic Basis of Disease. Philadelphia, WB Saunders, 1989, p 843 6. Evans DJ Jr, Evans DG, Graham DY, Klein PD: A sensitive and specific serologic test for detection of Campylobacter pytori infection. Gastroenterology 96:1004-1008, 1989 7. Dwyer B, Kaldor J, Tee W, Raios K: The prevalence of Campylobacter pylori in human populations. In Campylobacterpylori and Gastroduodenal Disease. BJ Rathbone, RV Heatley (eds). Oxford, Blackwell Scientific Publications, 1989, pp 190-196 8. Dwyer B, Nanxiong S, Kaldor J, Tee W, Lambert J, Luppino M, Flannery G: Antibody response to Campylobacter pylori in an ethnic group lacking peptic ulceration. Scand J Infect Dis 20:63-68, 1988 9. Jones DM, Eldridge J, Fox AJ, Sethi AJ, Whorwell PJ: Antibody to the gastric campylobacter-like organism ("Campylobacter pylori")---clinical correlations and distribution in the normal population. J Med Microbiol 22:57-62, 1986 10. Kosunen TU, Hook J, Rautelin HI, Myllyla G: Agedependent increase of Campylobacter pylori antibodies in blood donors. Scand J Gastroenterol 24:110-114, 1989 11. AI-Moagel MA, Evans DG, Abdulghani ME, Adams E, Evans DJ Jr, Malaty HM, Graham DY: Prevalence of Helicobacter (formally Campylobacter) pylori infection in Saudi Arabia and comparison of those with and without upper gastrointestinal symptoms. Am J Gastroenterol 85:944-948, 1990
10 8 8
12. Graham DY, Adam E, Klein PD, Evans DG, Evans DJ Jr, Alpert LC, Yoshimura HH, Brown M, Michaletz PA: Comparison of the prevalence of asymptomatic C. pylori infection in the United States: effect of age, gender and race. Gastroenterology 96:A180, 1989 13. Klein PD, The Gastrointestinal Physiology Working Group of Cayetano Heredia, and The Johns Hopkins Universities, Graham DY, Opekun AR, Skeley S, Evans DG, Evans DJ Jr: High prevalence of Campylobacter pylori (CP) infection in poor and rich Peruvian children determined by 13C urea breath test (13C-UBT). Gastroenterology 96:A260, 1989 14. Mitchell HM, Lee A, Berkowicz J, Barody T: The use of serology to diagnose active Campylobacter pylori infection. Med J Aust 149:604-609, 1988 15. Megraud F, Brassens-Rebbe M-P, Denis F, Belbouri A, Hoa DQ: Seroepidemiology of C. pylori infection in various populations. J Clin Microbiol 27:1870-1873, 1989 16. Morris A, Nicholson G, Lloyd G, Haines D, Rogers A, Taylor D: Seroepidemiology of Campylobacter pylori. NZ Med J 99:657-659, 1986 17. Vira D, D'Anastasio CD, Holton J, Dowsett JF, Londei M, Bertoni F, Beltrandi E, Grauenfels P, Salmon PR, Gandolfi L: Campylobacter pylori in abattoir workers: Is it a zoonosis. Lancet 2:725-726, 1988 18. Dwyer B, Kaldor J, Tee W, Marakowski E, Raios K: Antibody response to Campylobacter pylori in diverse ethnic groups. Scand J Infect Dis 20:394-350, 1988 19. Alpert LC, Graham DY, Evans DJ, Jr, Yoshimura HH, Hazell SL, Evans DG, Klein PD: Diagnostic possibilities for Campylobacter pylori infection. Eur J Gastroenterol Hepatol 1:17-26, 1989 20. Paul JR: Historical and geographical aspects of the epidemiology of poliomyelitis. Yale J Biol Med 27:101-113, 1954 21. Paul JR. A History of Poliomyelitis. New Haven, Yale University Press, 1971 22. Walton M, Melnick JL: Poliomyelitis antibodies in two differing socioeconomic groups within the same city. Yale J Biol Med 27:350-370, 1955 23. Dienstag JL, Szmuness W, Stevens CE, Purcell RH: Hepatitis A virus infection: new insights from seroepidemiologic studies. J Infect Dis 137:328-340, 1978 24. Frosner GG, Papaevangelou G, Butler R, Iwarson S, Lindholm A, Courouce-Pauty A, Haas H, Deinhardt F: Antibody against hepatitis A in seven European countries. I. Comparison of prevalence data in different age groups. Am J Epidemiol 110:93-99, 1979 25. Hsu HY, Chang MH, Chen DS, Lee CY, Sung JL: Changing seroepidemioiogy of hepatitis A virus infection in Taiwan. J Med Virol 17:297-301, 1985 26. Frosner G, Wilers H, Muller R, Schenzle D, Deinhardt F, Hopken W: Decrease in incidence of hepatitis A infections in Germany. Infection 6:259-260, 1978 27. Perez-Perez GI, Taylor DN, Bodhidatta L, Wongsrichanalai J, Baze WB, Dunn BE, Escheverria PD, Blaser MJ: Seroprevalence of H. pylori infections in Thailand. J Infect Dis 161:1237-1241, 1990
Digestive Diseases and Sciences, Vol. 36, No. 8 (August 1991)
Seroepidemiology of Helicobacter pylori Infection in India Comparison of Developing and Developed Countries DAVID Y. GRAHAM, ERVIN ADAM, GURUNATH T. REDDY, JAI PRAKASH AGARWAL, ROHIT AGARWAL, DOYLE J. EVANS, JR., HODA M. MALATY, and DOLORES G. EVANS
Helicobacter pylori (previously Campylobacter pylori) is now accepted as the major cause of type B gastritis and thus what is known about the epidemiology of type B gastritis can reasonably be transferred to H. pylori. We used a specific ELISA for anti-H, pylori lgG to study the prevalence ofH. pylori infection in a population of lower socioeconomic class from Hyderabad, India. The results from India were compared to studies from other parts of the world. Two hundred thirty-eight individuals ages 3 to 70 participated. The frequency ofH. pylori infection increased with age (P < 0.01) and was >80% by age 20. H. pylori infection was present in 79% of the population studied; there was no gender-related difference in prevalence of H. pylori infection. IgG antibo~' against hepatitis A (HA V) was rapidly acquired in Hyderabad; in a subset of 58 children between the ages o f 3 and 21 tested, the frequency of anti-HA V was 98.2%. The prevalence of H. pylori infection increases with age in both developed and developing countries. The high age-specific prevalence of H. pylori infection in developing countries is probably a reflection of the lower socioeconomic level of those areas. KEY WORDS: Helicobacter pylori; seroepidemiology; race; age-specific; socioeconomic group; hepatitis A.
Helicobacter pylori (previously Campylobacter pylori) is now accepted as the major cause of type B gastritis and thus what is known about the epidemiology of type B gastritis can reasonably be transferred to H. pylori. For example, we know that gastritis is more frequent in those of low socioeconomic class, large families, and crowding, and varManuscript received July 11, 1990; revised manuscript received January 22, 1991; accepted January 28, 1991. From the Department of Medicine and Division of Molecular Virology, Veterans Affairs Medical Center and Baylor College of Medicine, Houston, Texas; and Deccan Pathological Institute, Osmania General Hospital, and the Niloufer Hospital, Hyderabad, India. This work was supported by the research funds from the Department of Veterans Affairs; by grant DK 39919 from the National Institute of Diabetes and Digestive and Kidney Diseases, and by the generous support of Hilda Schwartz. Address for reprint requests: Dr. David Y. Graham, V. A. Medical Center (111D), 2002 Holcombe Blvd., Houston, Texas 77030.
1084
ies depending on the ethnic population studied. Ethanol, nicotine, and nonsteroidal antiinflammatory drug use do not play an important role (1-4). The incidence of gastritis varies both between and within countries (5). H. pylori infections are worldwide. Most reports have emphasized H. pylori infections in patients presenting for endoscopy and thus provide little information about the frequency in the general population. Others have used sera obtained from blood donors, which are often very select groups. We used a sensitive and specific ELISA to detect anti-H, pylori IgG antibodies against purified highmolecular-weight cell-associated antigens (HMCAP) to evaluate the prevalence of H. pylori infection (6). We also compared the rate of acquisition of H. pylori antibody with that of antibody to hepatitis A. We report the prevalence of H. pylori infection Digestive Diseases and Sciences, Vol. 36, No. 8 (August 1991)
0163-2I16/91/0800-1084506,50/09 1991PlenumPublishingCorporation
SEROEPIDEMIOLOGY OF Helicobacter pylori INFECTION
in a population of lower socioeconomic class from Hyderabad, India, and compare these results to studies f r o m o t h e r parts of the world.
M A T E R I A L S AND M E T H O D S Hyderabad is a cosmopolitan city in southern India with a population of approximately 4 million. Adults. The plan was to obtain serum specimens from approximately 20 individuals per age group. The study populations were obtained in July and August, 1988, from a walk-in outpatient medical clinic of the Osmania General Hospital, Hyderabad, India. Patients with any serious illness such as chronic heart, liver, renal, or lung disease were excluded. Patients were selected who (1) presented for minor illnesses or for follow-up during two days of the week (Tuesday and Thursday), (2) were within the age range(s) of the study, and (3) agreed to participate. Each patient was questioned concerning drug use, smoking, and symptoms referable to the upper gastrointestinal tract. Children. The study populations were obtained in February and March, 1989, from an outpatient pediatric clinic of Niloufer Hospital of the Institute of Pediatrics, Hyderabad, India. Children were selected who were within the age range(s) of the study, and parental consent was obtained. Both hospitals are public hospitals providing charity care to the lower socioeconomic class. Each patient had a blood sample obtained; the serum was separated and frozen until transfer to Houston, Texas. ELISA for Anti-H. pylori IgG. Specific details are published elsewhere (6); briefly, high-molecular-weight cell-associated protein obtained from n-octyl-glucoside extract of H. pylori cells was placed on an Agarose A-5m column and eluted with 0.05 M Tris C1 buffer pH 8.0. The two or three fractions possessing maximum urease activity were pooled. These fractions contain proteins in the molecular weight range of 400,000 to 700,000. The final antigen preparation contained at least two proteins, and analysis of ELISA-positive sera has demonstrated antibody against both of these major proteins. The ELISA was done in 96-well Microtiter plates (Linbro Scientific Co., Hamden, Connecticut) coated with HM-CAP diluted with PBS to approximately 7 ixg/ml. Sera diluted 1 to 50 and 1 to 100 were added and alkaline phosphatase-conjugated anti-human IgG (Southern Biotechnology, Birmingham, Alabama) was used as the detector. Each plate included HM-CAP ELISApositive and -negative control sera. The specificity and positive predictive value of the HM-CAP ELISA of the test were each shown to be 100%; the sensitivity is 98.7% and the negative predictive value 98.6%. There is no cross-reactivity with C. jejuni (unpublished studies). Antibody to hepatitis A (IgG) was determined on a random sample of 58 enrollees age 2-20 years. Antihepatitis A IgG was measured using the Abbott HAVABEIA assay. Data Analyses. The data was analyzed by logistic regression and Mantel-Haenszel chi square (SAS Institute, Digestive
Diseases
and
Sciences,
Vol. 36, No.
8 (August
1991)
TABLE 1. DISTRIBUTION OF THE STUDY POPULATION BY AGE AND SEX
Age range
Number
Male
Female
3-10 11-15 16-20 21-30 31-40 41-50 51-60 61-70
30 18 21 59 50 33 17 10
14 12 14 38 32 21 14 6
16 6 7 21 18 12 3 4
Cary, North Carolina). Age was a control variable in all analyses done. RESULTS
Two hundred thirty-eight individuals participated (Table 1). The male-female ratio was 1.7 to I. The frequency of H. pylori infection increased with age (P < 0.01) and was >80% by age 20 (Figure 1). There was no difference in prevalence of H. pylori infection between males and females (79% for both). Thirty-eight enrollees whose age was >20 had symptoms referable to the upper gastrointestinal tract; these symptoms were not the reason for visiting the outpatient clinic. The frequency of H. pylori infection in those with symptoms (84%) was the same as those without symptoms referable to the upper gastrointestinal tract (84%). There also was no difference in the frequency of H. pylori infection relating to whether the enrollee was a smoker or not. IgG antibody against hepatitis A (HAV) was rapidly acquired in Hyderabad. In 58 individuals 100 C.9
-r__ o Q..
80
-~ 6O
ON i
I
9
9
n =
238
~ 40
~ 2o n
o
...,
0
.....
10
, .....
20
, .....
,
.....
30 40 Age
,
. . . .
50
,
. . . . .
60
,
70
Fig 1. The age-specific acquisition of antibody to H. pylori infection is shown. The age ranges plotted are 3-10, 11-15, 16-20, 21-30, 31-40, 41-50, 51-60, and 61-70. 1 085
GRAHAM ET AL TABLE 2. SUMMARYOF SEROEPIDEMIOLOGICSTUDIES OF H. pylori INFECTION IN DEVELOPING AND DEVELOPED POPULATIONS Percent with antibodies to H. pylori (N in group) Age group Country
Australia Ref 18 Ref 14 Ref 7, 8 New Guinea Ref 14 Ref 14 New Zealand Ref 16 France Ref 15 England Ref 9 El Salvador Ref 18 Ethiopia Ref 18 Vietnam Ref 18
Population studied
10-19
20-29
30-39
40-49
50-59
Il (19) 5 (58) 0 (106) 59 (22) 20 33 (165) 16 (98) 4 (238) 43 (14)
0 (32) 18 (167) 0 (80) 66 (44) 38 45 (93) 25 (105) 19 (64) 25 (16) 47 (17) 12 (49) 75 (92) 75 (78) 85 (83) 75 (100) 81 (59)
13 (24) 14 (253) 0 (43) 58 (26) 28 54 (35) 27 (229) 19 (53) 47 (17) 41 (46) 22 (51) 78 (72) 83 (40) 88 (49) 73 (85) 92 (50)
17 (36) 27 (188) 0 (27) 75 (12) 54 33 (12) 33 (285) 16 (37) 50 (16) 24 (21) 17 (47) 85 (50) 75 (22) 95 (27) 75 (64) 88 (33)
30 (33) 37 (94) 11 (18) 64 (11) 40 0 (3) 35 (324) 40 (28) 33 (12)
White blood donors Unspecified, white?
4 (25)
Aborigines Papua, unadsorbed Papua, adsorbed Unspecified, blood donors? Bordeaux, outpatients Mixed
36 (112) 32 (175) 4 (113) 5 (22)
Immigrants to Australia Immigrants to Australia Immigrants to Australia Vietnam, blood donors
Ref 15 Ivory Coast Ref 15 Algeria Ref 15 Saudia Arabia Ref 11 India This study
0-9
Not specified Blood donors, outpatients Community-based healthy subjects Outpatients
13 (61) 55 (116) 45 (42) 42 (65) 60 (30)
45 (43) 75 (I00) 75 (59) 45 (! 37) 69 (39)
between the ages of 3 and 21, the frequency of anti-HAV was 98.2%. DISCUSSION
Published studies on the epidemiology of H.
pylori infection show considerable differences in the prevalence of antibodies to H. pylori in different populations studied (2, 7-18). The interpretation of these findings and the possible association of the infection with later sequelae of H. pylori exposure has its limitations due to two major factors: the representation of the population group studied, and the laboratory methods used for detection of specific antibodies to H. pylori (19). Nevertheless, a certain common pattern allows us to draw necessary further working hypotheses. The specimen collections that resulted in the data shown in Tables 2 and 3 include samples from different sources such as blood donors, newcomers to a given country, persons visiting outpatient health clinics for reasons other than gastrointestinal 1086
22 (18) 52 (21) 70 (7) 88 (17) 85 (53) 100 (17)
>60
20 (10)
33 (45) 47 (165)
24 (25) 55 (26) 80 (I1) 80 (45) 80 (10)
Totals
15 (154) 20 (785) 0.7 (277) 56 (157) 36 (483) 25 (1199) 21 (607) 40 (75) 43 (74) 18 (190) 53 (365) 73 (374) 79 (277) 72 (490) 81 (238)
symptoms, and from people presenting for regular periodic check-ups. It is not clear whom these groups represent, and socioeconomic differences and different exposures to infection are highly probable. Moreover, these serum collections include TABLE 3. PREVALENCEOF ANTIBODY TO H. pylori IN DIFFERENT POPULATION GROUPS IN NEW ZEALAND* Groups studied
Ethnic groups Maori Cook Island Samoan Tongan Occupational groups South Island meat inspectors Freezing works, veterinary surgeons Auckland meat workers Pig farmers Dairy farmers Pest control officers
Studied (N)
Infected (%)
192 85 129 56
21 39 44 70
18
17
18 80 68 20 14
44 41 13 20 21
*From reference 16. Digestive Diseases and Sciences. Vol. 36, No. 8 (August 1991)
SEROEPIDEMIOLOGY OF Helicobacter pylori INFECTION different ethnic groups with a prevalence ranging from 21% to 70%, such as in New Zealand (Table 3). Outpatient clinics in some countries may represent a mostly indigent population. Health-conscious persons visiting well-established health centers and high socioeconomic groups are probably not represented in any geographic area shown in Table 2. One problem in interpretation of data relates to the assays used to identify an H. pylori infection. The first-generation serologic tests employing whole bacteria, bacterial sonicates, or extracts of whole bacteria have all suffered from a high frequency of false positive tests caused by the presence of antibodies directed against antigen of C. jejuni or other bacteria containing antigens that cross-react with H. pylori (14, 19). The presence of cross-reacting antibodies appears to be particularly severe in studies using sera obtained from individuals living in developing countries. Recently, the suggestion was made that serologic tests should be restandardized for each population (14). Nevertheless, there are common patterns in t h e studies shown in Table 2. Evidently, the acquisition of H. pylori infection starts early in life and continues for several decades. Although the prevalence rates may be substantially different because of selection biases or methodological faults, the age-dependent trend is preserved. The difference in the prevalence ofH. pylori antibodies between groups also remains when samples are tested in the same laboratory. For example, the blood donors in Australia differ substantially from a group of Aborigines who were virtually not exposed to H. pylori infection and from ulcer patients who have a significantly higher rate of H. pylori antibodies--independent of age. Another example of sera tested in the same laboratory comes from France where blood donors in Vietnam or Algeria and the Ivory Coast had a substantially higher prevalence of antibodies to H. pylori than the local French population (15). Finally, Vietnamese who immigrated to Australia had H. pylori antibody rates significantly lower than blood donors in Vietnam. It is open to speculation if this difference is related to the assay per se or if there is a substantial socioeconomic difference between the local Vietnamese population represented by blood donors and emigrants. Despite the complexity of comparison of data from different geographic areas, the data support the concept that in some populations one major pathway of transmission is fecal-oral. The data also support the notion that the age-specific prevalence Digestive Diseases and Sciences, Vol. 36, No. 8 (August 1991)
of H. pylori infection, expressed as IgG antibody to H. pylori or those obtained using the [13C] urea breath test in Peru (13), in developing countries is related epidemiologically to the generally socioeconomic level of the geographic area. For example, Australian Aborigines showed two patterns: southern Aborigines had a higher rate of acquisition of H. pylori antibody than white Australians, as would be expected in a population of low socioeconomic status. In contrast, northern Aborigines appear to be a closed community in which H. pylori has not been introduced. The emerging pattern of the epidemiology of H. pylori infections is very reminiscent of the epidemiology of two other fecal-oral transmitted diseases, poliomyelitis and hepatitis A. For example, until the last century, poliomyelitis had always been a mild or subclinical disease in infants and as immunity is lifelong, older children and adults were immune. Improvements in the public environment, personal hygiene, sanitation, housing, and education resulted in an increasingly larger portion of the population that escaped exposure during infancy (20-22). This resulted in an even larger population of susceptible individuals in whom the disease would manifest as a paralytic poliomyelitis. The change to epidemics of paralytic poliomyelitis prompted a number of excellent epidemiologic studies proving an inverse relationship between the rate of acquisition of anti-polio antibody and standards of hygiene. In populations with poor standards, the steepest rise in antibody prevalence occurred in the youngest age group, whereas those with higher standards (eg, directly related to socioeconomic levels) experienced a delay with the steepest point of the curve in children of school age or adolescence. The differences in the prevalence of paralytic poliomyelitis were not based on geography per se but were related to standards and practices of hygiene and socioeconomic conditions. The epidemiology of hepatitis A was found to mirror that of poliomyelitis (23-26). It appears that the same factors that affected the age-specific prevalence of antibody to poliomyelitis (before vaccination) and to HAV also predict the age-specific prevalence of antibody to H. pylori. HAV is much more contagious than H. pylori and thus the rate of acquisition is more rapid and serves as a marker for poor sanitary conditions (11, 27). We postulate that, if both HAV and H. pylori were transmitted by the fecal-oral route, a similar shift in the age of acquisition of H. pylori infection could also be demon-
1087
GRAHAM ET AL s t r a t e d in the s a m e p o p u l a t i o n . C o n s e q u e n t l y , a shift in the o n s e t o f H. pylori i n f e c t i o n c o u l d d e l a y the o n s e t o f d u o d e n a l ulcer d i s e a s e a n d result in a r e d u c t i o n in the f r e q u e n c y o f gastric c a r c i n o m a b y d e l a y i n g the o n s e t o f the p r e c u r s o r lesion, a t r o p h i c gastritis.
ACKNOWLEDGMENTS The authors thank F. B. Hollinger for performing the hepatitis A antibody determinations.
REFERENCES 1. Graham DY, Adam E, Klein PD, Evans DJ Jr, Evans DG, Hazell SL, Alpert LC, Michaletz PA, Yoshimura HH: Epidemiology of Carnpylobacter pylori. Gastroenterol Clin Biol 13:84B-88B, 1989 2. Graham DY, Klein PD, Opekun AR, Boutton TW, et al: Epidemiology of Campylobacter pylori infection: Ethnic considerations. Scand J Gastroenterol 23 (suppl 142): 9-13, 1988 3. Massarrat S, Paidlik A, Pittner P, Schmitz-Moorman P, Wurbs M: The role of certain habits and various diseases in the occurrence of gastritis. Hepato-Gastroenterol 30:249, 1983 4. Edwards FC, Goghill NF: Aetiological factors in chronic atrophic gastritis. Br Med J 2:1409-1415, 1966 5. Cotran RS, Kumar V, Robbins SL (eds): Robbins Pathologic Basis of Disease. Philadelphia, WB Saunders, 1989, p 843 6. Evans DJ Jr, Evans DG, Graham DY, Klein PD: A sensitive and specific serologic test for detection of Campylobacter pytori infection. Gastroenterology 96:1004-1008, 1989 7. Dwyer B, Kaldor J, Tee W, Raios K: The prevalence of Campylobacter pylori in human populations. In Campylobacterpylori and Gastroduodenal Disease. BJ Rathbone, RV Heatley (eds). Oxford, Blackwell Scientific Publications, 1989, pp 190-196 8. Dwyer B, Nanxiong S, Kaldor J, Tee W, Lambert J, Luppino M, Flannery G: Antibody response to Campylobacter pylori in an ethnic group lacking peptic ulceration. Scand J Infect Dis 20:63-68, 1988 9. Jones DM, Eldridge J, Fox AJ, Sethi AJ, Whorwell PJ: Antibody to the gastric campylobacter-like organism ("Campylobacter pylori")---clinical correlations and distribution in the normal population. J Med Microbiol 22:57-62, 1986 10. Kosunen TU, Hook J, Rautelin HI, Myllyla G: Agedependent increase of Campylobacter pylori antibodies in blood donors. Scand J Gastroenterol 24:110-114, 1989 11. AI-Moagel MA, Evans DG, Abdulghani ME, Adams E, Evans DJ Jr, Malaty HM, Graham DY: Prevalence of Helicobacter (formally Campylobacter) pylori infection in Saudi Arabia and comparison of those with and without upper gastrointestinal symptoms. Am J Gastroenterol 85:944-948, 1990
10 8 8
12. Graham DY, Adam E, Klein PD, Evans DG, Evans DJ Jr, Alpert LC, Yoshimura HH, Brown M, Michaletz PA: Comparison of the prevalence of asymptomatic C. pylori infection in the United States: effect of age, gender and race. Gastroenterology 96:A180, 1989 13. Klein PD, The Gastrointestinal Physiology Working Group of Cayetano Heredia, and The Johns Hopkins Universities, Graham DY, Opekun AR, Skeley S, Evans DG, Evans DJ Jr: High prevalence of Campylobacter pylori (CP) infection in poor and rich Peruvian children determined by 13C urea breath test (13C-UBT). Gastroenterology 96:A260, 1989 14. Mitchell HM, Lee A, Berkowicz J, Barody T: The use of serology to diagnose active Campylobacter pylori infection. Med J Aust 149:604-609, 1988 15. Megraud F, Brassens-Rebbe M-P, Denis F, Belbouri A, Hoa DQ: Seroepidemiology of C. pylori infection in various populations. J Clin Microbiol 27:1870-1873, 1989 16. Morris A, Nicholson G, Lloyd G, Haines D, Rogers A, Taylor D: Seroepidemiology of Campylobacter pylori. NZ Med J 99:657-659, 1986 17. Vira D, D'Anastasio CD, Holton J, Dowsett JF, Londei M, Bertoni F, Beltrandi E, Grauenfels P, Salmon PR, Gandolfi L: Campylobacter pylori in abattoir workers: Is it a zoonosis. Lancet 2:725-726, 1988 18. Dwyer B, Kaldor J, Tee W, Marakowski E, Raios K: Antibody response to Campylobacter pylori in diverse ethnic groups. Scand J Infect Dis 20:394-350, 1988 19. Alpert LC, Graham DY, Evans DJ, Jr, Yoshimura HH, Hazell SL, Evans DG, Klein PD: Diagnostic possibilities for Campylobacter pylori infection. Eur J Gastroenterol Hepatol 1:17-26, 1989 20. Paul JR: Historical and geographical aspects of the epidemiology of poliomyelitis. Yale J Biol Med 27:101-113, 1954 21. Paul JR. A History of Poliomyelitis. New Haven, Yale University Press, 1971 22. Walton M, Melnick JL: Poliomyelitis antibodies in two differing socioeconomic groups within the same city. Yale J Biol Med 27:350-370, 1955 23. Dienstag JL, Szmuness W, Stevens CE, Purcell RH: Hepatitis A virus infection: new insights from seroepidemiologic studies. J Infect Dis 137:328-340, 1978 24. Frosner GG, Papaevangelou G, Butler R, Iwarson S, Lindholm A, Courouce-Pauty A, Haas H, Deinhardt F: Antibody against hepatitis A in seven European countries. I. Comparison of prevalence data in different age groups. Am J Epidemiol 110:93-99, 1979 25. Hsu HY, Chang MH, Chen DS, Lee CY, Sung JL: Changing seroepidemioiogy of hepatitis A virus infection in Taiwan. J Med Virol 17:297-301, 1985 26. Frosner G, Wilers H, Muller R, Schenzle D, Deinhardt F, Hopken W: Decrease in incidence of hepatitis A infections in Germany. Infection 6:259-260, 1978 27. Perez-Perez GI, Taylor DN, Bodhidatta L, Wongsrichanalai J, Baze WB, Dunn BE, Escheverria PD, Blaser MJ: Seroprevalence of H. pylori infections in Thailand. J Infect Dis 161:1237-1241, 1990
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