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Bactericidal antibody and susceptibility to otitis media caused by nontypable strains of Haemophilus influenzae
364
September 1980 TheJournalofPED1ATRICS
Bactericidal antibody and susceptibility to otitis media caused by nontypable strains of Haemophilus
influenzae We have developed a bactericidal antibody assay to determine the role o f circulating antibody to nontypable strains ofHaemophilus influenzae in children with otitis media. Antibody to the infecting strain was present in the acute sera o f 2/20 children with otitis media caused by H. influenzae NT,. 15/18 o f these patients had convalescent specimens with titers >~ 1:2 (X ~ = 13.0, P < 0.001). The acute sera of 95 children with otitis media caused by various organisms were screened for bactericidal activity against a randomly selected H. influenzae N T strain. Antibody was present in 0/28 acute sera of patients whose infection was caused by H. influenzae N T and in 18/67 (26.9%) o f those whose middle ear exudates were sterile or contained other bacterial species (X z = 9.95, P < 0.01). A bactericidal antibody response generally follows infection of the middle ear with H. influenzae NT. The absence of antibody to a single strain in the sera o f some children appears to be associated with susceptibility to this infection.
Paul A. Shurin, M.D.,* Stephen I. Pelton, M.D., Ira B. Tager, M.D., and Dennis L. Kasper, M.D.,** Boston, Mass.
THE HIGH ATTACK RATE for otitis media in infancy suggests that acquired immunity may protect older individuals. The complement-mediated bactericidal antibody From the Channing Laboratory and the Departments of Pediatrics and Medicine, Harvard Medical School, the Departments o f Pediatrics and Medical Microbiology, Boston City Hospital, and the Department o f Pediatrics, Boston University School of Medicine. Supported by a grant (HD03693) from the National Institute o f Child Health and Human Development and by Contracts NO1-HR32906 and NO1-HR32944 from the National Heart, Lung and Blood Institute. Prepared in honor o f Dr. Edward H. Kass and has been presented, in part, at a Program on Infectious Diseases and Related Topics hem on the occasion o f his sixtieth birthday, and to the 17th Interscience Conference on Antimicrobial Agents and Chemotherapy, New York, October 13, 197Z *Reprint address: Department of Pediatrics, Cleveland Metropolitan General Hospital, 3395 Scranton Rd, Cleveland, OH 44109. **Recipient of Career Development A ward 1 K04 A1 00126 from the National Institute of All~?rgy and Infectious Diseases
Vol. 97, No.3, pp. 364-369
reaction has potential functional significance in immunity to gram-negative infections.1 Since strains of H a e m o p h i l u s influenzae which are not typable according to the Pittman classification are important agents of this infection,2'we have studied the role of serum bactericidal antibody to H. influenzae NT in otitis media. Abbreviations used NT: nontypable cfu: colony-forming units To: time of Onset T:,o: termination
METHODS Subjects. The patients constituted a consecutively studied series of 118 children with otitis media, 2 months to 12 years of age? After parental consent was obtained, the diagnosis of otitis media was confirmed by transtympanie aspiration of the middle-ear effusions. Blood specimens were obtained at entry into the study and at follow-up visits. Aspiration and culture of middle-ear effusions were repeated when indicated clinically.
0022-3476/80/090364+06500.60/0 9 1980 The C. V. Mosby Co.
Volume 97 Number 3
Bacterial strains. Thirty strains of H. influenzae NT were obtained by culture of middle-ear effusions. The bacteria were identified as H. influenzae by colonial morphology and requirement for factors X and V, and as NT by failure to give capsular swelling with antisera to types a-f obtained from the Center for Disease Control (Atlanta). Bactericidal assay. Organisms. Two-tenths milliliter of an overnight growth of the H. influenzae strain to be tested was inoculated into Levinthal broth 4 and grown to a concentration of 2 • 10~ cfu/ml in the logarithmic phase of growth as determined by optical density. For use in the bactericidal assay, the test organisms were diluted to a final dilution of 2 • 104 cfu/ml in a solution of Gey balanced salt solution (Grand Island Biological Company, Grand Island, NY) and 10% (v/w) bovine serum albumin (Cohn Fraction V, Sigma Chemical Company, St. Louis). Sera. Blood specimens were obtained by venipuncture from the human subjects and by cardiac puncture from a rabbit immunized with a viable H. influenzae NT strain (BCH strain T 1). Sera were stored at - 2 0 C. Sera obtained during antimicrobiat drug administration were not used in these studies. Complement. Preliminary studies demonstrated that the human volunteer serum that was obtained for use as a complement source contained bactericidal activity against H. influenzae NT. To remove this activity, the serum was absorbed ~ with whole cells of four selected 1t. influenzae NT strains. Aliquots of absorbed complement were stored at -70~ thawed immediately before use in each experiment, and not re-used. The complement activity of the absorbed serum was compared with that of fresh-frozen serum of a subject with agammaglobulinemia. The absorbed and agammaglobulinemic sera had hemolytic activities of 62.5 and 71.0 CH~0 units/ml, respectively. These sera were also compared as complement sources in parallel bactericidal assays of the acute and convalescent sera of four patients with otitis media and their homologous isolates of H. influenzae. In all of these assays the results obtained with the two complement sources were not different from each other, using the methods and criteria specified below. Therefore, the bactericidal tests reported below were performed using 0.05 ml of the absorbed serum as the complement source. This volume was shown to be in excess of the volume needed in the bactericidal reaction. Procedure for bactericidal assay. The bactericidal assay used in these experiments was a modification of that used by Kasper, et al ~ for Neisseria gonorrhoeae. The reaction mixture consisted of 0.075 ml of test serum diluted in Gey
H. influenzae and otitis media
36 5
solution to give dilutions of 1:2 to 1:16 in the final reaction mixture, 0.05 ml complement, and 0.025 ml of the bacterial suspension. Immediately after addition of the bacterial suspension to each tube, a 0.025 ml aliquot was removed and spread with a glass rod on the surface of a chocolate agar plate. The tubes were then incubated at 37~ for 30 minutes in a rotary-shaker water bath. At the end of this incubation period, duplicate 0.025 ml aliquots were spread on chocolate agar plates. After overnight incubation of the plates, colonies were counted for determination of the viable cfu at the onset (To) and termination (T30) of the incubation period. The mean of counts in the duplicate aliquots was taken as the value for T30. Controls for each bactericidal assay consisted of separate tubes containing bacterial suspension and the following reactants: (1) active complement and Gey solution without test serum; (2) heat-inactivated complement (56~ for 30 minutes) and serum; and (3) heat-inactivated complement and 0.075 ml of heat inactivated test serum. In addition, each assay included a tube with heatinactivated immune rabbit serum diluted to its endpoint, the homologous immunizing H. influenzae strain (BCH strain T 0 and complement. Many of the stored serum specimens, particularly those with high titer antibody, contained residual complement activity and thus gave killing when tested with heat-inactivated complement. Significant bacterial killing as defined below was not obtained in the controls containing complement alone or in those in which both serum and complement were inactivated. The majority of control tubes gave an increase in cfu during the incubation period. Thus, the patient sera did not have bactericidal activity other than that mediated by the complement-dependent reaction. All tests showed killing by the immune rabbit serum, demonstrating both good reproducibility and adequacy of the complement used for each assay. Bactericidal assays of acute and convalescent sera of patients with otitis media caused by H. influenzae NT were performed using each patient's homologous H. influenzae NT isolate and serial twofold dilutions (1:2 to 1:16) of that patient's sera. Prozone phenomena were not observed with any of the sera tested. Screening of acute sera of patients with otitis media was performed using a 1:2 final dilution of each serum tested against a single randomly selected H. influenzae NT strain (BCH 37567). This test strain had induced an antibody response in the patient from whom it had been isolated. Data analysis. The results of the bactericidal assays were evaluated using both the percentage of the initial inoculum which was killed and the absolute reduction in cfu of the inoculum after 30 minutes' incubation. The
366
Shurin et al.
The Journal of Pediatrics September 1980
0 -Otitis media not caused by non-typable H. influenzae
100 -
9 -Otitis media caused by non-typable H. influenzae
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Figure. Bactericidal activity against H. influenzae NT strain 37567 of acute sera of 95 children with otitis media.
Reduction in viable counts observed during 30-minute incubation period using 0.075 ml serum, 0.05 ml absorbed human complement, and 0.025 ml bacterial suspension. Serum specimens used were obtained at the same time as the cultures of middle ear exudates.
percentage of the inoculum killed at 30 minutes was defined as: cfu% - cfu%0 x 100. cfu% For evaluation of patients with otitis media and their homologous H. influenzae N T strains, significant bactericidal activity was arbitrarily defined as killing of > 90% of the starting inoculum. The titers of acute and convalescent sera were defined as the highest dilution of each which gave this 90% reduction. For those convalescent sera Which did not exceed a titer of 1:2 (i.e., not a fourfold increase in titer) the difference between corresponding acute and convalescent specimens in their absolute reduction of the initial inoculum was evaluated by paired t test. 6 The initial inocula for seven such pairs of sera did not differ significantly from each other (t6 = 0.68, P > 0.5). When the criterion of 90% reduction was applied to the sera screened for bactericidal activity against 1t. influenzae strain 37567, killing of this magnitude was infrequently observed even at a 1:2 dilution (Figure). However, it was observed that a substantial n u m b e r of the subjects had a 50% reduction of the initial inoculum. Therefore, since this latter criterion has been employed for other population studies of bactericidal antibody to H. influenzae, 7, ~ analysis of the screening data employed the 50% endpoint. Two by two contingency tables were a n a l y z J t using the M c N e m a r chi square test for paired dataY
RESULTS Patients with otitis media due to H. influenzae NT.
Each patient's sera were assayed for bactericidal activity against the H. influenzae N T strain which had been obtained from the same patient's middle ear exudate (Table I). The sera from 18 of 20 patients (90%) obtained at or before presentation with ofitis media had titers < 1:2. Two patients had titers equal to 1:2 in these sera. Of 18 patients who returned for follow-up (11 to 264 days after presentation), 15 had at least one serum specimen with a titer _> 1:2 (X2 = 13.0, P < 0.001). Seven of these follow-up specimens had triers of 1:2. The reduction in T~0 cfu by these seven specimens was significantly greater than given by the corresponding acute sera, with a m e a n difference of 70 cfu/O.025 ml" (t~ = 7.60, P < 0.001; 95% confidence interval 52 to 88 cfu). In each of six patients for whom early ( < 30 days) and late (_> 31 days) convalescent sera were available, the later serum specimen showed greater percentage killing than the early specimen. The two patients with initial titers of 1:2 had convalescent titers of 1:8 and 1 :>_ 16, respectively. (Individual patient data available from authors upon request). Prevalence of bactericidal antibody to H. influenzae strain 37567 in acute sera o f patients with otitis media o f
varying etiology. Acute sera were available from 95 of the entire series of 118 children with otitis media. At entry
Volume 97 Number 3 into the study, bacterial isolates had been obtained from the middle ear effusions of 69 patients (H. influenzae NT = 28; Streptococcus pneumoniae = 29; Neisseria catarrhalis = 5; Staphylococcus aureus and H. influenzae type b = 2 each; and H. influenzae type f, N. meningitidis and Streptococcus pyogenes = 1 each). Middle ear effusions were sterile or contained nonpathogenic bacteria in 26 patients. Patients with otitis media due to H. influenzae NT were comparable to the other patients in terms of age (median 1.1 years and 1.0 years, respectively), sex (50% and 51% males), race (39% and 48% black), and history of prior episodes of otitis media (median one episode per patient in each group). None of the acute sera from patients with otitis media due to H. influenzae NT had bactericidal antibody (as defined by 50% killing of the bacterial inoculum, c.f. Methods) against strain BCH 37567 (Figure). In contrast, bactericidal antibody was present in 18 of 67 acute sera (26.9%) from patients whose otitis media was not due to H. influenzae NT (X2 = 9.95, P < 0.01). Our investigation of episodes of otitis media which occurred after entry into the study was not sufficiently complete to establish the frequency or immunologic correlations of subsequent middle ear infection due to H. influenzae. Subsequent episodes of otitis media due to H. influenzae NT were, however, observed only in children who had initially lacked bactericidal antibody to strain 37567 (Table II). Four of the five children who experienced such recurrent infection had had H. influenzae NT isolated from middle ear exudates during the presenting episode of otitis media.
H. influenzae and otitis media
367
Table I. Serum bactericidal antibody activity against homologous bacterial isolates in children with otitis media caused by nontypable strains of Haemophilus influenzae*
, Time of serum collection Before infection Acute Convalescent
,Recipr~ titer'~
Number of patients <21214 8 6 18 3
l 2 7
0 0 3
>--16
0 0 1
0 0 4
*Sera were assayed against the strain of H. influenzae isolated from the same patient's middle ear effusion. tReciprocal of highest serum dilution giving _> 90% reduction in viable bacterial count.
Table II. Recurrent otitis media in study patients; Isolation of NT strains of IL influenzae from recurrent middle ear exudates according to presence of bactericidal antibody at entry into study No. of patients with recurrent otitis media due to H . i n f l u e n z a e NT/No. of patients with etiologically defined recurrent episodes*
Etiology of index episode H. influenzae NT Other bacteria or nonbacterial
Bactericidal antibody to strain 37567t +
0 0/7
4/14 1/19
*Episodes of otitis media occurring ~> 7 days after entry into the trial and defined microbiologically by culture of middle ear exudates. "~Presence of antibody defined by killing of _> 50% of the bacterial inoculum.
DISCUSSION
Haemophilus influenzae NT has been isolated from middle ear effusions of approximately 20% of cases of acute otitis mediaY Serum antibody responses to this infection have been documented with bactericidal,TM complement fixation,11-~4 and fluorescent antibody1~ assays, but the possible protective role of circulating antibody has not previously been assessed. The present study has demonstrated that lack of circulating bactericidal antibody to H. influenzae NT is associated with infection of the middle ear by this organism. Children with this infection did not have antibody to the test strain at the time of diagnosis. In contrast, antibody was significantly more likely to be present in sera of the control children. Antigenic diversity among H. influenzae NT strains is well known14"6; the present results suggest that the test strain used may contain antigenic determinants which induce protective antibody. The nature of such antigens is unknown at present.
Our tests of individual patients' sera showed that most lacked detectable antibody to the infecting strains of H. influenzae NT at the time of diagnosis or prior to infection. The majority of these children had a convalescent bactericidal antibody response to infection. There was no apparent correlation of age with the ability to respond; infants as young as three months had convalescent antibody. This fact may be contrasted with the relative inability of infants to respond to the capsular antigen of H. influenzae type b following colonization, immunization, or natural infection.'7 Immunization with noncapsular antigens might thus be of value in protecting against disease due to type b or nontypable strains. We have not determined the mechanism of antibodymediated protection against otitis media. The antibody detected by our test may reflect a host defense acting by a different mechanism than bacterial lysis. Findings in patients with immunodeficiency support a possible role
368
Shurin et al.
for complement-dependent i m m u n e defenses in otitis media. Patients with agammaglobulinemia TM or with specific defects of complement components is, 20 have been particularly susceptible to this disease. There are several possible explanations for the lack of antibody found in acute sera of our patients with otitis media due to H. influenzae NT. They may have lacked prior immunizing experience with the organism or with antigens of the infecting strains. Alternatively, blocking antibodies might have inhibited bactericidal activity at the time of infection. 21 Finally, it is possible that o u r patients with otitis media due to H. influenzae N T had circulating bacterial antigens which inhibited bactericidal activity in acute serum specimens. Antigenic inhibition seems a relatively unlikely explanation for the findings in acute sera of otitis media patients since bactericidal activity was lacking both in sera obtained prior to the onset of H. influenzae N T infection and in the acute sera of most of the group of age-matched otitis m e d i a patients who did not have infection due to this organism. Nasopharyngeal carriage of H. influenzae N T is highly prevalent in young children and all children become colonized at some time. 22 Antibody directed against H. influenzae N T strains might protect by reducing colonization, as occurs in the presence of pneumococca128 and meningococcal anticapsular antibody, ~4 b y preventing invasion of the middle ear, 2~ or by promoting clearance and recovery from otitis. ~ It is likely, however, that deficiency o f specific antibody is not the sole determinant of middle ear infection. Other factors which are thought to contribute to the prevalence of middle ear disease in children are physiologic dysfunction of the eustachian tube, racial or genetic factors, adverse patterns of infant feeding, and viral infections of the respiratory tract. We are indebted to Dr. Robertson Parkman, Children's Hospital Medical Center, Boston, for supplying the agammaglobulinemic serum and to Diana Moore, Carlos Fernandez, and Joan Finkelstein for technical assistance. Bacterial strains were isolated and identified in the Laboratory of Medical Microbiology of the Boston City Hospital by A. Kathleen Daly and Alice McDonald. REFERENCES
t. Goldschneider, I, Gotschlich, EC, Artenstein, MS: Human immunity to the meningococcus. 1. The role of humoral antibodies, J Exp Med 129:1307, 1969. 2. Harding AL, Anderson P, Howie VM, Ploussard JH, and Smith DH: Hemophilus inftuenzae isolated from children with otitis media, in Sell SHW, and Karzon DT: Hemophilus influenzae, Nashville, Tenn., 1973, Vanderbilt University Press, p 21. 3. Shurin PA, Pelton SI, Donner A, Finkelstein J, and Klein JO: Trimethoprim-sulfamethoxazole compared with ampi-
The Journal of Pediatrics September i980
cillin in the treatment of acute otitis media, J Pediatr 96:1081, 1980. 4. McLinn SE, Nelson JD, and Haltalin KC: Antimicrobial susceptibility of Hemophilus influenzae, Pediatrics 45:827, 1970.
5. Kasper DL, Rice PA, and McCormack WM: Bactericidal antibody in genital infection due to Neisseria gonorrhoeae, J Infect Dis 135:243, 1977. 6. Armitage P: Statistical methods in medical research, New York, 1971, John Wiley & Sons Inc. 7. Anderson P, Johnston RB Jr, and Smith DH: Human serum activities against Hemophilus influenzae, type b, J Clin Invest 51:31, 1972. 8. Norden CW: Prevalence of Bactericidal Antibodies to Haemophilus influenzae, type b , J infect Dis 130:489, 1974. 9. Bluestone CD, and Shurin PA: Middle ear disease in children; pathogenesis, diagnosis, and management, Pediatr Clin North Am 2i:379, 1974. 10. Sloyer JL Jr, Cate CC, Howie VM, Ploussard JH, and Johnston RB Jr: The immune response to acute otitis media in children. II. Serum and middle ear fluid antibody in otitis media due to Haemophilus influenzae, J Infect Dis 132:685, 1975. 11. Bjuggren G, and Tunevall G: The Pfeiffer bacillus in otitis in children: A sero-bacteriological and clinical study, Acta Otolaryngol, 38:130, 1950. 12. Bjuggren G, and Tunevall G: Otitis in childhood: A clinical and sero-bacteriological study with special reference to the significance of Haemophilus influenzae in relapses, Acta Otolaryngol 42:311, 1952. 13. Branefors-Helander P, Nyl6n O, and Jeppsson P-H: Acute otitis media. Assay of complement-fixing antibody against Haemophilus influenzae as a diagnostic tool in acute otitis media, Acta Pathol M{crbbiol Scand Section B 81:508, 1973. 14. Tunevall G: Studies on Haemophilus influenzae: A complement fixation test for Haemophilus inlluenzae antibody, Acta Pathol Microbiol Scand 32:259, 1953. 15. Tunevalt G: Studies on Haemophilus influenzae: Haemophilus influenzae antigens studied by th e gel precipitation method, Acta Pathol Microbiol Scand 32:193, 1953. 16. May JR: Colonial morphology, antigens and pathogenicity of Haemophilus influenzae, in The scientific basis of medicine annual reviews, London, 1967, The Athlone Press p 211. 17. Anderson P, Smith DH, Ingram DL, Wilkins J, Wehrle PF, and Howie VM: Antibody to pOlyribophosphate of Haemophilus influenzae type b in infants and children: Effect of immunization with polyribophosphate, J Infect Dis 136(Suppl):S57; 1977. 18. Rosen FS, and Janeway CA: The gamma globulins. III. The antibody deficiency syndromes, N Engl J Med 275:709 and 769, 1966. 19. Alper CA, Abramson N, Johnston RB Jr, Jandl JH, and Rosen FS: Increased susceptibility to infection associated with abnormalities of complement-mediated functions and of the third component of complement (C3), N Engl J Med 282:349, 1970. 20. Rynes RI, Urizar RE, and Pickering R J: Genetic deficiency of the second component of complement (C2) associated
Volume 97 Number 3
with systemic lupus erythematosus: Relation of the complement abnormality and disease manifestations, Am J Med 63:279, 1977. 21. McCutchan JA, Katzenstein D, Norquist D, Chikami G, Wunderlich A, and Braude AI: Role of blocking antibody in disseminated gonococcal infection, J Immunol 121:1884, 1978. 22. Kilian M, Heine-Jensen J, and Btilow P: Haemophilus in the upper respiratory tract of children, Acta Path Microbiol Stand Sect B 80:571, 1972. 23. MacLeod CM, Hodges RG, Heidelberger M, and Bernhard WG: Prevention of pneumococcal pneumonia by immunization with specific capsular polysaccharides, J Exp Med 82:445, 1945.
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24. Artenstein MS, Gold R, Zimmerly JG, Wyle FA, Schneider H, and Harkins C: Prevention of meningococcal disease by group C polysaccharide vaccine, N Engl J Med 282:417, 1970. 25. Giebink GS, Berzins IK, Schiffman G, and Quie PG: Experimental otitis media in chinchillas following nasal colonization with type 7F Streptococcus pneumoniae: Prevention.after vaccination with pneumococcal capsular polysaccharide, J Infect Dis 140:716, 1979. 26. Sloyer JL Jr, Howie VM, Ploussard JH, Schiffman G, and Johnston RB Jr: Immune response to otitis media: association between middle ear fluid antibody and clearing of clinical infection, J Clin Microbiol 4:306, 1976.
September 1980 TheJournalofPED1ATRICS
Bactericidal antibody and susceptibility to otitis media caused by nontypable strains of Haemophilus
influenzae We have developed a bactericidal antibody assay to determine the role o f circulating antibody to nontypable strains ofHaemophilus influenzae in children with otitis media. Antibody to the infecting strain was present in the acute sera o f 2/20 children with otitis media caused by H. influenzae NT,. 15/18 o f these patients had convalescent specimens with titers >~ 1:2 (X ~ = 13.0, P < 0.001). The acute sera of 95 children with otitis media caused by various organisms were screened for bactericidal activity against a randomly selected H. influenzae N T strain. Antibody was present in 0/28 acute sera of patients whose infection was caused by H. influenzae N T and in 18/67 (26.9%) o f those whose middle ear exudates were sterile or contained other bacterial species (X z = 9.95, P < 0.01). A bactericidal antibody response generally follows infection of the middle ear with H. influenzae NT. The absence of antibody to a single strain in the sera o f some children appears to be associated with susceptibility to this infection.
Paul A. Shurin, M.D.,* Stephen I. Pelton, M.D., Ira B. Tager, M.D., and Dennis L. Kasper, M.D.,** Boston, Mass.
THE HIGH ATTACK RATE for otitis media in infancy suggests that acquired immunity may protect older individuals. The complement-mediated bactericidal antibody From the Channing Laboratory and the Departments of Pediatrics and Medicine, Harvard Medical School, the Departments o f Pediatrics and Medical Microbiology, Boston City Hospital, and the Department o f Pediatrics, Boston University School of Medicine. Supported by a grant (HD03693) from the National Institute o f Child Health and Human Development and by Contracts NO1-HR32906 and NO1-HR32944 from the National Heart, Lung and Blood Institute. Prepared in honor o f Dr. Edward H. Kass and has been presented, in part, at a Program on Infectious Diseases and Related Topics hem on the occasion o f his sixtieth birthday, and to the 17th Interscience Conference on Antimicrobial Agents and Chemotherapy, New York, October 13, 197Z *Reprint address: Department of Pediatrics, Cleveland Metropolitan General Hospital, 3395 Scranton Rd, Cleveland, OH 44109. **Recipient of Career Development A ward 1 K04 A1 00126 from the National Institute of All~?rgy and Infectious Diseases
Vol. 97, No.3, pp. 364-369
reaction has potential functional significance in immunity to gram-negative infections.1 Since strains of H a e m o p h i l u s influenzae which are not typable according to the Pittman classification are important agents of this infection,2'we have studied the role of serum bactericidal antibody to H. influenzae NT in otitis media. Abbreviations used NT: nontypable cfu: colony-forming units To: time of Onset T:,o: termination
METHODS Subjects. The patients constituted a consecutively studied series of 118 children with otitis media, 2 months to 12 years of age? After parental consent was obtained, the diagnosis of otitis media was confirmed by transtympanie aspiration of the middle-ear effusions. Blood specimens were obtained at entry into the study and at follow-up visits. Aspiration and culture of middle-ear effusions were repeated when indicated clinically.
0022-3476/80/090364+06500.60/0 9 1980 The C. V. Mosby Co.
Volume 97 Number 3
Bacterial strains. Thirty strains of H. influenzae NT were obtained by culture of middle-ear effusions. The bacteria were identified as H. influenzae by colonial morphology and requirement for factors X and V, and as NT by failure to give capsular swelling with antisera to types a-f obtained from the Center for Disease Control (Atlanta). Bactericidal assay. Organisms. Two-tenths milliliter of an overnight growth of the H. influenzae strain to be tested was inoculated into Levinthal broth 4 and grown to a concentration of 2 • 10~ cfu/ml in the logarithmic phase of growth as determined by optical density. For use in the bactericidal assay, the test organisms were diluted to a final dilution of 2 • 104 cfu/ml in a solution of Gey balanced salt solution (Grand Island Biological Company, Grand Island, NY) and 10% (v/w) bovine serum albumin (Cohn Fraction V, Sigma Chemical Company, St. Louis). Sera. Blood specimens were obtained by venipuncture from the human subjects and by cardiac puncture from a rabbit immunized with a viable H. influenzae NT strain (BCH strain T 1). Sera were stored at - 2 0 C. Sera obtained during antimicrobiat drug administration were not used in these studies. Complement. Preliminary studies demonstrated that the human volunteer serum that was obtained for use as a complement source contained bactericidal activity against H. influenzae NT. To remove this activity, the serum was absorbed ~ with whole cells of four selected 1t. influenzae NT strains. Aliquots of absorbed complement were stored at -70~ thawed immediately before use in each experiment, and not re-used. The complement activity of the absorbed serum was compared with that of fresh-frozen serum of a subject with agammaglobulinemia. The absorbed and agammaglobulinemic sera had hemolytic activities of 62.5 and 71.0 CH~0 units/ml, respectively. These sera were also compared as complement sources in parallel bactericidal assays of the acute and convalescent sera of four patients with otitis media and their homologous isolates of H. influenzae. In all of these assays the results obtained with the two complement sources were not different from each other, using the methods and criteria specified below. Therefore, the bactericidal tests reported below were performed using 0.05 ml of the absorbed serum as the complement source. This volume was shown to be in excess of the volume needed in the bactericidal reaction. Procedure for bactericidal assay. The bactericidal assay used in these experiments was a modification of that used by Kasper, et al ~ for Neisseria gonorrhoeae. The reaction mixture consisted of 0.075 ml of test serum diluted in Gey
H. influenzae and otitis media
36 5
solution to give dilutions of 1:2 to 1:16 in the final reaction mixture, 0.05 ml complement, and 0.025 ml of the bacterial suspension. Immediately after addition of the bacterial suspension to each tube, a 0.025 ml aliquot was removed and spread with a glass rod on the surface of a chocolate agar plate. The tubes were then incubated at 37~ for 30 minutes in a rotary-shaker water bath. At the end of this incubation period, duplicate 0.025 ml aliquots were spread on chocolate agar plates. After overnight incubation of the plates, colonies were counted for determination of the viable cfu at the onset (To) and termination (T30) of the incubation period. The mean of counts in the duplicate aliquots was taken as the value for T30. Controls for each bactericidal assay consisted of separate tubes containing bacterial suspension and the following reactants: (1) active complement and Gey solution without test serum; (2) heat-inactivated complement (56~ for 30 minutes) and serum; and (3) heat-inactivated complement and 0.075 ml of heat inactivated test serum. In addition, each assay included a tube with heatinactivated immune rabbit serum diluted to its endpoint, the homologous immunizing H. influenzae strain (BCH strain T 0 and complement. Many of the stored serum specimens, particularly those with high titer antibody, contained residual complement activity and thus gave killing when tested with heat-inactivated complement. Significant bacterial killing as defined below was not obtained in the controls containing complement alone or in those in which both serum and complement were inactivated. The majority of control tubes gave an increase in cfu during the incubation period. Thus, the patient sera did not have bactericidal activity other than that mediated by the complement-dependent reaction. All tests showed killing by the immune rabbit serum, demonstrating both good reproducibility and adequacy of the complement used for each assay. Bactericidal assays of acute and convalescent sera of patients with otitis media caused by H. influenzae NT were performed using each patient's homologous H. influenzae NT isolate and serial twofold dilutions (1:2 to 1:16) of that patient's sera. Prozone phenomena were not observed with any of the sera tested. Screening of acute sera of patients with otitis media was performed using a 1:2 final dilution of each serum tested against a single randomly selected H. influenzae NT strain (BCH 37567). This test strain had induced an antibody response in the patient from whom it had been isolated. Data analysis. The results of the bactericidal assays were evaluated using both the percentage of the initial inoculum which was killed and the absolute reduction in cfu of the inoculum after 30 minutes' incubation. The
366
Shurin et al.
The Journal of Pediatrics September 1980
0 -Otitis media not caused by non-typable H. influenzae
100 -
9 -Otitis media caused by non-typable H. influenzae
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o
0 -
o
9
c~ 0
o " 2
.6
10
I 15 PATIENT
-T~" : 20 40 AGE
I 80
I 120
Oo I 160
(Months)
Figure. Bactericidal activity against H. influenzae NT strain 37567 of acute sera of 95 children with otitis media.
Reduction in viable counts observed during 30-minute incubation period using 0.075 ml serum, 0.05 ml absorbed human complement, and 0.025 ml bacterial suspension. Serum specimens used were obtained at the same time as the cultures of middle ear exudates.
percentage of the inoculum killed at 30 minutes was defined as: cfu% - cfu%0 x 100. cfu% For evaluation of patients with otitis media and their homologous H. influenzae N T strains, significant bactericidal activity was arbitrarily defined as killing of > 90% of the starting inoculum. The titers of acute and convalescent sera were defined as the highest dilution of each which gave this 90% reduction. For those convalescent sera Which did not exceed a titer of 1:2 (i.e., not a fourfold increase in titer) the difference between corresponding acute and convalescent specimens in their absolute reduction of the initial inoculum was evaluated by paired t test. 6 The initial inocula for seven such pairs of sera did not differ significantly from each other (t6 = 0.68, P > 0.5). When the criterion of 90% reduction was applied to the sera screened for bactericidal activity against 1t. influenzae strain 37567, killing of this magnitude was infrequently observed even at a 1:2 dilution (Figure). However, it was observed that a substantial n u m b e r of the subjects had a 50% reduction of the initial inoculum. Therefore, since this latter criterion has been employed for other population studies of bactericidal antibody to H. influenzae, 7, ~ analysis of the screening data employed the 50% endpoint. Two by two contingency tables were a n a l y z J t using the M c N e m a r chi square test for paired dataY
RESULTS Patients with otitis media due to H. influenzae NT.
Each patient's sera were assayed for bactericidal activity against the H. influenzae N T strain which had been obtained from the same patient's middle ear exudate (Table I). The sera from 18 of 20 patients (90%) obtained at or before presentation with ofitis media had titers < 1:2. Two patients had titers equal to 1:2 in these sera. Of 18 patients who returned for follow-up (11 to 264 days after presentation), 15 had at least one serum specimen with a titer _> 1:2 (X2 = 13.0, P < 0.001). Seven of these follow-up specimens had triers of 1:2. The reduction in T~0 cfu by these seven specimens was significantly greater than given by the corresponding acute sera, with a m e a n difference of 70 cfu/O.025 ml" (t~ = 7.60, P < 0.001; 95% confidence interval 52 to 88 cfu). In each of six patients for whom early ( < 30 days) and late (_> 31 days) convalescent sera were available, the later serum specimen showed greater percentage killing than the early specimen. The two patients with initial titers of 1:2 had convalescent titers of 1:8 and 1 :>_ 16, respectively. (Individual patient data available from authors upon request). Prevalence of bactericidal antibody to H. influenzae strain 37567 in acute sera o f patients with otitis media o f
varying etiology. Acute sera were available from 95 of the entire series of 118 children with otitis media. At entry
Volume 97 Number 3 into the study, bacterial isolates had been obtained from the middle ear effusions of 69 patients (H. influenzae NT = 28; Streptococcus pneumoniae = 29; Neisseria catarrhalis = 5; Staphylococcus aureus and H. influenzae type b = 2 each; and H. influenzae type f, N. meningitidis and Streptococcus pyogenes = 1 each). Middle ear effusions were sterile or contained nonpathogenic bacteria in 26 patients. Patients with otitis media due to H. influenzae NT were comparable to the other patients in terms of age (median 1.1 years and 1.0 years, respectively), sex (50% and 51% males), race (39% and 48% black), and history of prior episodes of otitis media (median one episode per patient in each group). None of the acute sera from patients with otitis media due to H. influenzae NT had bactericidal antibody (as defined by 50% killing of the bacterial inoculum, c.f. Methods) against strain BCH 37567 (Figure). In contrast, bactericidal antibody was present in 18 of 67 acute sera (26.9%) from patients whose otitis media was not due to H. influenzae NT (X2 = 9.95, P < 0.01). Our investigation of episodes of otitis media which occurred after entry into the study was not sufficiently complete to establish the frequency or immunologic correlations of subsequent middle ear infection due to H. influenzae. Subsequent episodes of otitis media due to H. influenzae NT were, however, observed only in children who had initially lacked bactericidal antibody to strain 37567 (Table II). Four of the five children who experienced such recurrent infection had had H. influenzae NT isolated from middle ear exudates during the presenting episode of otitis media.
H. influenzae and otitis media
367
Table I. Serum bactericidal antibody activity against homologous bacterial isolates in children with otitis media caused by nontypable strains of Haemophilus influenzae*
, Time of serum collection Before infection Acute Convalescent
,Recipr~ titer'~
Number of patients <21214 8 6 18 3
l 2 7
0 0 3
>--16
0 0 1
0 0 4
*Sera were assayed against the strain of H. influenzae isolated from the same patient's middle ear effusion. tReciprocal of highest serum dilution giving _> 90% reduction in viable bacterial count.
Table II. Recurrent otitis media in study patients; Isolation of NT strains of IL influenzae from recurrent middle ear exudates according to presence of bactericidal antibody at entry into study No. of patients with recurrent otitis media due to H . i n f l u e n z a e NT/No. of patients with etiologically defined recurrent episodes*
Etiology of index episode H. influenzae NT Other bacteria or nonbacterial
Bactericidal antibody to strain 37567t +
0 0/7
4/14 1/19
*Episodes of otitis media occurring ~> 7 days after entry into the trial and defined microbiologically by culture of middle ear exudates. "~Presence of antibody defined by killing of _> 50% of the bacterial inoculum.
DISCUSSION
Haemophilus influenzae NT has been isolated from middle ear effusions of approximately 20% of cases of acute otitis mediaY Serum antibody responses to this infection have been documented with bactericidal,TM complement fixation,11-~4 and fluorescent antibody1~ assays, but the possible protective role of circulating antibody has not previously been assessed. The present study has demonstrated that lack of circulating bactericidal antibody to H. influenzae NT is associated with infection of the middle ear by this organism. Children with this infection did not have antibody to the test strain at the time of diagnosis. In contrast, antibody was significantly more likely to be present in sera of the control children. Antigenic diversity among H. influenzae NT strains is well known14"6; the present results suggest that the test strain used may contain antigenic determinants which induce protective antibody. The nature of such antigens is unknown at present.
Our tests of individual patients' sera showed that most lacked detectable antibody to the infecting strains of H. influenzae NT at the time of diagnosis or prior to infection. The majority of these children had a convalescent bactericidal antibody response to infection. There was no apparent correlation of age with the ability to respond; infants as young as three months had convalescent antibody. This fact may be contrasted with the relative inability of infants to respond to the capsular antigen of H. influenzae type b following colonization, immunization, or natural infection.'7 Immunization with noncapsular antigens might thus be of value in protecting against disease due to type b or nontypable strains. We have not determined the mechanism of antibodymediated protection against otitis media. The antibody detected by our test may reflect a host defense acting by a different mechanism than bacterial lysis. Findings in patients with immunodeficiency support a possible role
368
Shurin et al.
for complement-dependent i m m u n e defenses in otitis media. Patients with agammaglobulinemia TM or with specific defects of complement components is, 20 have been particularly susceptible to this disease. There are several possible explanations for the lack of antibody found in acute sera of our patients with otitis media due to H. influenzae NT. They may have lacked prior immunizing experience with the organism or with antigens of the infecting strains. Alternatively, blocking antibodies might have inhibited bactericidal activity at the time of infection. 21 Finally, it is possible that o u r patients with otitis media due to H. influenzae N T had circulating bacterial antigens which inhibited bactericidal activity in acute serum specimens. Antigenic inhibition seems a relatively unlikely explanation for the findings in acute sera of otitis media patients since bactericidal activity was lacking both in sera obtained prior to the onset of H. influenzae N T infection and in the acute sera of most of the group of age-matched otitis m e d i a patients who did not have infection due to this organism. Nasopharyngeal carriage of H. influenzae N T is highly prevalent in young children and all children become colonized at some time. 22 Antibody directed against H. influenzae N T strains might protect by reducing colonization, as occurs in the presence of pneumococca128 and meningococcal anticapsular antibody, ~4 b y preventing invasion of the middle ear, 2~ or by promoting clearance and recovery from otitis. ~ It is likely, however, that deficiency o f specific antibody is not the sole determinant of middle ear infection. Other factors which are thought to contribute to the prevalence of middle ear disease in children are physiologic dysfunction of the eustachian tube, racial or genetic factors, adverse patterns of infant feeding, and viral infections of the respiratory tract. We are indebted to Dr. Robertson Parkman, Children's Hospital Medical Center, Boston, for supplying the agammaglobulinemic serum and to Diana Moore, Carlos Fernandez, and Joan Finkelstein for technical assistance. Bacterial strains were isolated and identified in the Laboratory of Medical Microbiology of the Boston City Hospital by A. Kathleen Daly and Alice McDonald. REFERENCES
t. Goldschneider, I, Gotschlich, EC, Artenstein, MS: Human immunity to the meningococcus. 1. The role of humoral antibodies, J Exp Med 129:1307, 1969. 2. Harding AL, Anderson P, Howie VM, Ploussard JH, and Smith DH: Hemophilus inftuenzae isolated from children with otitis media, in Sell SHW, and Karzon DT: Hemophilus influenzae, Nashville, Tenn., 1973, Vanderbilt University Press, p 21. 3. Shurin PA, Pelton SI, Donner A, Finkelstein J, and Klein JO: Trimethoprim-sulfamethoxazole compared with ampi-
The Journal of Pediatrics September i980
cillin in the treatment of acute otitis media, J Pediatr 96:1081, 1980. 4. McLinn SE, Nelson JD, and Haltalin KC: Antimicrobial susceptibility of Hemophilus influenzae, Pediatrics 45:827, 1970.
5. Kasper DL, Rice PA, and McCormack WM: Bactericidal antibody in genital infection due to Neisseria gonorrhoeae, J Infect Dis 135:243, 1977. 6. Armitage P: Statistical methods in medical research, New York, 1971, John Wiley & Sons Inc. 7. Anderson P, Johnston RB Jr, and Smith DH: Human serum activities against Hemophilus influenzae, type b, J Clin Invest 51:31, 1972. 8. Norden CW: Prevalence of Bactericidal Antibodies to Haemophilus influenzae, type b , J infect Dis 130:489, 1974. 9. Bluestone CD, and Shurin PA: Middle ear disease in children; pathogenesis, diagnosis, and management, Pediatr Clin North Am 2i:379, 1974. 10. Sloyer JL Jr, Cate CC, Howie VM, Ploussard JH, and Johnston RB Jr: The immune response to acute otitis media in children. II. Serum and middle ear fluid antibody in otitis media due to Haemophilus influenzae, J Infect Dis 132:685, 1975. 11. Bjuggren G, and Tunevall G: The Pfeiffer bacillus in otitis in children: A sero-bacteriological and clinical study, Acta Otolaryngol, 38:130, 1950. 12. Bjuggren G, and Tunevall G: Otitis in childhood: A clinical and sero-bacteriological study with special reference to the significance of Haemophilus influenzae in relapses, Acta Otolaryngol 42:311, 1952. 13. Branefors-Helander P, Nyl6n O, and Jeppsson P-H: Acute otitis media. Assay of complement-fixing antibody against Haemophilus influenzae as a diagnostic tool in acute otitis media, Acta Pathol M{crbbiol Scand Section B 81:508, 1973. 14. Tunevall G: Studies on Haemophilus influenzae: A complement fixation test for Haemophilus inlluenzae antibody, Acta Pathol Microbiol Scand 32:259, 1953. 15. Tunevalt G: Studies on Haemophilus influenzae: Haemophilus influenzae antigens studied by th e gel precipitation method, Acta Pathol Microbiol Scand 32:193, 1953. 16. May JR: Colonial morphology, antigens and pathogenicity of Haemophilus influenzae, in The scientific basis of medicine annual reviews, London, 1967, The Athlone Press p 211. 17. Anderson P, Smith DH, Ingram DL, Wilkins J, Wehrle PF, and Howie VM: Antibody to pOlyribophosphate of Haemophilus influenzae type b in infants and children: Effect of immunization with polyribophosphate, J Infect Dis 136(Suppl):S57; 1977. 18. Rosen FS, and Janeway CA: The gamma globulins. III. The antibody deficiency syndromes, N Engl J Med 275:709 and 769, 1966. 19. Alper CA, Abramson N, Johnston RB Jr, Jandl JH, and Rosen FS: Increased susceptibility to infection associated with abnormalities of complement-mediated functions and of the third component of complement (C3), N Engl J Med 282:349, 1970. 20. Rynes RI, Urizar RE, and Pickering R J: Genetic deficiency of the second component of complement (C2) associated
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with systemic lupus erythematosus: Relation of the complement abnormality and disease manifestations, Am J Med 63:279, 1977. 21. McCutchan JA, Katzenstein D, Norquist D, Chikami G, Wunderlich A, and Braude AI: Role of blocking antibody in disseminated gonococcal infection, J Immunol 121:1884, 1978. 22. Kilian M, Heine-Jensen J, and Btilow P: Haemophilus in the upper respiratory tract of children, Acta Path Microbiol Stand Sect B 80:571, 1972. 23. MacLeod CM, Hodges RG, Heidelberger M, and Bernhard WG: Prevention of pneumococcal pneumonia by immunization with specific capsular polysaccharides, J Exp Med 82:445, 1945.
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24. Artenstein MS, Gold R, Zimmerly JG, Wyle FA, Schneider H, and Harkins C: Prevention of meningococcal disease by group C polysaccharide vaccine, N Engl J Med 282:417, 1970. 25. Giebink GS, Berzins IK, Schiffman G, and Quie PG: Experimental otitis media in chinchillas following nasal colonization with type 7F Streptococcus pneumoniae: Prevention.after vaccination with pneumococcal capsular polysaccharide, J Infect Dis 140:716, 1979. 26. Sloyer JL Jr, Howie VM, Ploussard JH, Schiffman G, and Johnston RB Jr: Immune response to otitis media: association between middle ear fluid antibody and clearing of clinical infection, J Clin Microbiol 4:306, 1976.