Scandinavian Journal of Infectious Diseases
ISSN: 0036-5548 (Print) 1651-1980 (Online) Journal homepage: http://www.tandfonline.com/loi/infd19
Studies on Branhamella Catarrhalis (Neisseria Catarrhalis) with Special Reference to Maxillary Sinusitis John-Erik Brorson, Alf Axelsson & Stig E. Holm To cite this article: John-Erik Brorson, Alf Axelsson & Stig E. Holm (1976) Studies on Branhamella Catarrhalis (Neisseria Catarrhalis) with Special Reference to Maxillary Sinusitis, Scandinavian Journal of Infectious Diseases, 8:3, 151-155, DOI: 10.3109/inf.1976.8.issue-3.05 To link to this article: http://dx.doi.org/10.3109/inf.1976.8.issue-3.05
Published online: 02 Jan 2015.
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Date: 25 April 2016, At: 18:37
Scand J Infect Dis 8: 151-155. 1976
Studies on Branhamella catarrhalis (Neisseria catarrhalis) with Special Reference to Maxillary Sinusitis JOHN-ERIK BRORSON, ALF AXELSSON and STIG E. HOLM
Downloaded by [UQ Library] at 18:37 25 April 2016
From the Institute of Medical Microbiology, the Department of Clinical Bacteriology, University of Goteborg, Goteborg, the Department of Otolaryngology, Lundby Medical Center, and the Department of Clinical Bacteriology, University of Umed. Umed, Sweden
ABSTRACT. Paired sera from 97 patients with acute maxillary sinusitis were examined regarding antibodies to Branhamella catarrhalis. Precipitating antibodies were demonstrated in almost all sera both from patients and from healthy blood donors. Complement-fixing(CF) antibodies to B. catarrhalis were present in sera from 25 of the 97 patients and in one of 20 healthy blood donors. The titres were low and the titre changes when present were of a small magnitude. CF antibodies were most commonly demonstrated in the younger age groups. The patients with demonstrable CF antibodies to B. catarrhalis did not differ from patients without antibodies regardingradiological appearance or healing during therapy. Strains of B. catarrhalis were all rapidly killed by normal human serum but not in heated sera. The strains could not multiplicate significantly at an oxygen tension correspondingto about half the atmospheric value. The possible significance of the serological and bacteriological findings is discussed.
INTRODUCTION Branhamella catarrhalis, formerly known as Neisseria catarrhalis, has its natural habitat in the upper respiratory tract of man. It has been isolated from middle ear exudates of children suffering from acute middle ear suppuration (9, 15), and it has been proposed that the bacterium should be regarded as pathogenic in this condition (15). Sporadic cases of meningitis and septicemia due to this microbe have also been reported (1 1, 19). It has also been isolated, often in mixed cultures, from maxillary secretions of patients suffering from acute maxillary sinusitis (2, 10, 16). Serological studies on maxillary sinusitis. have to our knowledge not included B. catarrhalis. This fact initiated the present study of the immune response to B. catarrhalis in patients suffering from acute maxillary sinusitis. MATERIALS AND METHODS Study group The study group consists of 97 patients attending the ENT department for maxillary sinusitis. All had radiological signs of acute maxillary sinusitis with mucous membrane
thickening and secretion. The radiological method for demonstration of acute maxillary sinusitis and the evaluation of treatment has been described before (8). Sinus aspirates for bacteriological studies were obtained from all patients by puncture of the maxillary sinus after topical anesthesia with 4% lindocain. These specimens were processed as described earlier (2). Serological studies Blood was drawn by venipuncture on the first examination at the ENT department and after 15 days. Serum was recovered by centrifugation and was kept at -20°C until processed. Sera collected during the study period from 20 healthy blood donors with matched age groups served as controls. Antigens for immunodiffusion (ID) analyses were prepared from 10 different B. catarrhalis strains freshly isolated from young children with nasopharyngeal infections. The bacteria were seeded on agar plates on an antigen-free medium (13), harvested after 20 hours’ incubation at 3TC, washed 3 times in phosphate buffered saline pH 7.0. The sediment was suspended in 2 volumes of the same buffer and desintegrated in a Mickle cell desintegrator for 1 X 5 min under coolirig conditions. The preparation was centrifuged for 30 min at 2OOOOxg, and the supernate used in the ID analyses. A similar antigen was prepared from 20 different strains of Neisseria gonorrhoeae. Antigen for complement fixation (CF). Part of the bacteria harvested from the plates (see above) were washed and finally heated to 56°C for 30 min. The bacteria Scand J Infect Dis 8
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Table I . Age and sex distribution Numbers within brackets denote number of patients within each group with demonstrable C F antibodies to B. catarrhalis Healthy blood donors (control group)
Study group Age (Y.)
Males
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c20 2 1-30 31 4 0 4 1-50 S5 I
8 (3) 9 (3) 8 (3)
2 5 (2) 32 (1 1)
Females
4 (2) (7)
19 17 17 8
(I) (3) (1)
65 (14)
Total
Males
12 (5) 28 (10) 25 (4) 19 (3) 13 (3) 97 (25)
2 2 2
employed for the C F studies were used in the highest concentration not giving anticomplementary activity. Similarly a N. gonorrhoeae suspension was prepared and analysed. ID analyses were performed by means of the microgelchamber method of Wadsworth (22). CF-analyses were made largely in accordance with Kabat and Mayer (14). The highest serum dilution giving less than 50% hemolysis was registered as the limit value. The serum dilutions started at 1 :7.5. Antisera. Rabbits were hyperimmunized by weekly intramuscular injections of 0.2 ml of the desintegrated B. catarrhalis and N. gonorrhoeae antigens respectively in complete Freund adjuvant. The immune response was followed by weekly bleedings. Reference system. To establish the antigenic pattern of the two Neisseria antigen preparations the two antigens for ID analyses were tested with their homologous rabbit hyperimmunsera in ID. Between 10 and 15 separate precipitation lines were detected in each system illustrating the multiplicity of immunogens in the antigen preparations. No cross reactions were found between the N. gonorrhoeae and B. catarrhalis precipitating antigens. Studies on B . catarrhalis Bacteria. Three strains of B. catarrhalis were used. They were all recent clinical isolates. They were identified according to Berger (4).
... .
0
0
7.5
15
30
Initial titre
Fig. 1 . Comparison between C F titres to B. catarrhalis registered initially and 15 days later in 25 persons suffering from acute maxillary sinusitis. Scand J Infect Dis 8
Females
Total
1
4 (1) 4 3 I
3 6 (1) 6 3 2
7
13 (1)
20 ( I )
1
Bacterial inoculum. In all experiments the bacterial inoculum consisted of cells grown for 5 hours in ox-broth on a rotary shaker waterbath at 37°C. Viable counts were performed by inoculating bloodagar plates with appropriate dilutions from the different systems. The plates were read after 48 hours at 37°C. Influence of serum. Blood was drawn from 2 healthy volunteers without evidence of upper respiratory tract infection the last 8 weeks. Serum was obtained by centrifugation and was kept at -20°C for a maximum period of 2 weeks before use. One portion of each serum was heated to 56°C for 30 min. The different serum samples were inoculated with cells of the 3 strains washed twice in physiologic saline. Viable counts were performed after 1.5,6 and 24 hours. Influence ofoxygen tension. The 3 strains were grown in 20 ml ox-broth in 300 ml indented flasks. Each flask was inoculated to contain about lo3 cells/ml. One flask was placed on a rotary water bath at 37°C. Three flasks of each strain were placed at 37°C in separate anaerobic jars which were evacuated to 300 mmHg. Viable counts were performed after 6, 24 and 48 hours.
RESULTS Of the 97 patients with radiological signs of acute maxillary sinusitis 25 had CF antibodies against B. catarrhalis (Table I). The titres were low and the titre changes were of a small magnitude (Fig. I). C F antibodies were most commonly demonstrated in the younger age groups. Only one serum from the control group demonstrated C F antibodies. With the exception of 6 patients, precipitating antibodies to B. catarrhalis were demonstrated in sera from the 97 patients suffering from acute maxillary sinusitis as well as in the control group. These few sera originated from persons of older age. All patients with CF antibodies had precipitating antibodies. Comparative ID analyses showed that patients and with precipitins had at least one common antibody. The
BI.anhamella catarrhalis and maxillary sinusitis /
,-f/C'
Viable count
-
lo8-
#
/
#
/
/
X
1.5
6
24lime
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Fig. 2. Influence of human serum on the growth of B. catarrhalis. -=normal human serum; - - - =human serum heated to 56°C for 30 min. Time is given in hours.
number of antibodies registered in different sera varied between I and 3. The outcome of the treatment was measured radiologically. The 25 patients with demonstrable C F antibodies to B. catarrhalis did not differ from the 72 patients without CF antibodies regarding radiological appearance of healing during identical therapy. The influence of serum on B. catarrhalis can be seen from Fig. 2. Normal human serum had a pronounced killing effect. All 3 strains investigated were killed in normal serum whereas the bacteria grew in serum heated to 56°C for 30 min. All unheated sera affected the 3 strains to a comparable degree. The oxygen tension had a marked influence on growth rate of B. catarrhalis (Fig. 3). Only slight multiplication of the bacteria was demonstrable at reduced oxygen tension. DISCUSSION There are two modes of establishing an assumed etiological association between microbes and infectious disease, i.e. demonstration of the organism and/or serological demonstration of a titre change during the course of the disease. Both methods have their disadvantages. In the case of sinusitis aspirated secretion must be obtained since the nasal flora reflects the sinus flora unreliably (2). Furth-' ermore, the transport time, the transport medium and the handling of the bacteriological samples also influence the bacteriological results. It is also possible that the microorganism inducing infection is no longer present when the sinus aspirate is analyzed. Serological examinations are more difficult to
153
perform in ordinary praxis and it is often hard to motivate the patient to return after 2-3 weeks for a second blood sample when the disease already has subsided, treated or untreated, to assure a titre change as the significant serologic diagnostic criterium. There is an obvious discrepancy between the numerous reports dealing with the bacteriology of maxillary sinusitis (La. 5, 10, 16, 17, 18, 21) and the low number of reports dealing with serology in this disease (20). Surprisingly, many of the patients (25 out of 97) had demonstrable C F antibodies to B. catarrhalis and in most of these patients a moderate change in titre was observed. The facts that C F antibodies with one exception were not observed in the control group and that titre changes were observed in many of the patients suffering from acute maxillary sinusitis suggest that the titre changes might be significant for an association with the disease. Precipitating antibodies were demonstrated in almost all sera both from patients suffering from acute maxillary sinusitis and in the control group. A possible explanation for this may be that precipitating antibodies reflect the fact that B. catarrhalis is a normal inhabitant of the upper respiratory tract and that most persons are continuously exposed to its antigens resulting in the emergence of precipitating antibodies in the same way as was earlier demonstrated for streptococcal antibodies (12). On the other hand C F antibodies are usually connected with an actual or recent infection and they are usually not demonstrable for more than some months after the disease as was clearly shown for Haemophilus influenzae (6). Bacteriological studies on maxillary sinusitis differ widely regarding the frequency with which B.
10'1
I
, 6
r
24
48 lime
Fig. 3 . Influence of oxygen tension on the growth of B. catarrhalis. -=cells grown at 760 mmHg; - - - =cells grown at 300 mmHg. Time is given in hours. Scand J Infect Dis 8
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catarrhalis has been isolated on culture (5, 10, 16, 17, 18, 21). Thus, the present serological study could indicate that the correlation between culture and serology regarding B. catarrhalis in acute maxillary sinusitis is poor. There may be many explanations to such a suggestion but it should be mentioned that a lack of correlation between cultural and serological findings exists for other bacteria too. A positive correlation between isolation of e.g. H. influenzae from the maxillary sinus and demonstration of C F antibodies to this microbe in the same patients is far from complete (20). Another explanation to the lack of correlation between isolation of B. catarrhalis and detection of C F antibodies implies the possibility that the antibody response detected against B. catarrhalis in these patients is a part of the defence mechanism against this microorganism. The present study has also shown that B . catarrhalis is markedly influenced by factors which might contribute to the fact that the microbe relatively seldom is isolated from maxillary contents of patients suffering from acute maxillary sinusitis. It is well known that B. catarrhalis does not grow under anaerobic conditions. The finding that B. catarrhalis is almost unable to multiplicate at an oxygen tension corresponding to about half the atmospheric value might be of some importance in explaining the fact that B. catarrhalis is seldom isolated from maxillary aspirates. It is e.g. known that the oxygen content in a maxillary sinus with impaired drainage rapidly decreases to a value corresponding to about half the atmospheric value (1). It is also a well known fact that ostial dysfunction is very common in acute maxillary sinusitis making low oxygen tension quite plausible. It was also found that normal serum rapidly killed strains of B. catarrhalis and that this killing effect of serum was abolished by heat inactivation. This could indicate that the killing might be due to a complement-demanding process. No attempt was made to elucidate the factors responsible for the killing, but this study has also shown that it is very common to find precipitating antibodies to B. catarrhalis in sera from healthy persons. Many bacterial species have been demonstrated in acute maxillary sinusitis. Some degree of correlation has been observed between infecting bacterial species and initial radiological appearance and healing during therapy (3). In this study it was found that neither the initial radiological appearance nor Scotid J Infect Dis 8
the healing process during therapy could be correlated to the presence of C F antibodies to B. catarrhalis. The demonstration of C F antibodies to a microbe often present in the vicinity of the maxillary sinus is no proof that the microbe in fact is incriminated in acute maxillary sinusitis. In a parallel study, antibodies to other bacteria generally thought to be of etiological significance in acute maxillary sinusitis were often demonstrated alone or in combination with C F antibodies to B . catarrhalis (7). However, in some cases with negative culture for bacteria the only positive serological finding was the demonstration of C F antibodies to B. catarrhalis. It remains to be elucidated whether this indicates that B . catarrhalis should be regarded as being of etiological significance in acute maxillary sinusitis. Antigenic analyses of B . catarrhalis have clearly indicated that this microorganism should not be included among the Neisseria species. Rather it belongs to the Moraxella group. It should be noted that the Moraxella group is not antigenically related to H. influenzae or pneumococci which are generally incriminated in acute maxillary sinusitis. This indicates that the observed C F antibodies probably should be regarded as specific and not due to cross reactions with the bacteria most commonly seen in maxillary sinusitis. ACKNOWLEDGEMENT The excellent technical assistance of Mrs Ingrid Wargskog is gratefully acknowledged.
REFERENCES 1. Aust, R. & Drettner, B.: Ventilatory studies of the
maxillary sinus. Int Rhino1 9: 69, 1971. 2. Axelsson, A. & Brorson, J.-E.: The correlation between bacteriological findings in the nose and maxillary sinus in acute maxillary sinusitis. Laryngoscope 83: 2003, 1973. 3. Axelsson, A. & Chidekel, N.: Symptomatology and bacteriology correlated to radiology in acute maxillary sinusitis. Acta Otolaryngol (Stockh) 74: 118, 1972. 4. Berger, U.: Die anspruchlosen Neisserien. Ergeb Mikrobiol36: 97, 1963. 5 . Bjorkwall, T.: Bacteriological examinations in maxillary sinusitis. Acta Otolaryngol (Stockh), Suppl. 83: 1, 1950. 6. Branefors-Helander, P.: Serological studies of Haemophilus influenzae with special reference to otitis media infections. Dissertation, University of Goteborg, Sweden 1973.
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7. Brorson, J.-E., Axelsson, A. & Holm, S. E.: Serological studies in acute maxillary sinusitis. To be published. 8. Chidekel, N., Jensen, L., Axelsson, A. & Grebelius, N.: Diagnosis of fluid in the maxillary sinus. Acta Radio1 (Stockh) 10: 433, 1970. 9. Coffey, J. D., Martin, A. D. & Booth, H. N.: Neisseria catarrhalis in exudate otitis media. Arch Otolaryngol86: 403, 1967. 10. van Dishoeck, H. E. A. & Franssen, M. G. C.: The incidence and correlation of allergy and chronic maxillary sinusitis. Pract Otolaryngol 19: 502, 1957. I I . Elston, H. R., Quigley, H. J. & Fitch, D. M.: Meningitis due to Neisseria catarrhalis. Nebr State Med J 55: 369, 1970. 12. Hanson, L.-A. & Holm, S.: Precipitating antibodies in human sera from different age groups and in colostrum as determined by streptococcal antigens with diffusion-in-gel methods. Acta Paediatr Scand 50: 7, 1961. 13. Holm, S. E. & Falsen, E.: An antigen-free medium for cultivation of beta-hemolytic streptococci. Acta Path01 Microbiol Scand 69: 264, 1967. 14. Kabat, E. A. & Mayer, M. M.: Experimental immunochemistry, p. 133. Ch. C. Thomas, Springfield, Ill. 1967. 15. Kamme, C.: The aetiology of acute otitis media in children and the relationship between bacterial characters and the clinical course in penicillin V therapy. Dissertation, University of Lund, Sweden 1971. 16. Kinnman, J., Lee, C. H. & Park, S. H.: Bacterial flora in chronic, purulent maxillary sinusitis. Acta Otolaryngol (Stockh) 64: 37, 1967. 17. Kortekangas, A. E.: Antibiotics in the treatment of maxillary sinusitis. Acta Otolaryngol (Stockh), Suppl. 188: 379, 1963. 18. Lystad, A., Berdal, P. & Lund-Iversen, L.: The bacterial flora of sinusitis with an in vitro study of the bacterial resistance to antibiotics. Acta Otolaryngol (Stockh), Suppl. 188: 390, 1964. 19. Noguchi, T. T., Nachum, R. & Lawrence, C. A.: Acute purulent meningitis caused by chromogenic Neisseria. Med Arts Sci 17: 11, 1963. 20. Nylkn, O., Jeppsson, P.-H. & Branefors-Helander, P.: Acute sinusitis. A clinical, bacteriological and serological study with special reference to Haemophilus influenzae. Scand J Infect Dis 4: 43, 1972. 21. Piquet, J., Houcke, E. & Leclerq, J.: Les sinusites maxillares chroniques amicrobiennes. Ann Otolaryngo1 73:915, 1956. 22. Wadsworth, C.: A microplate technique employing a gel-chamber compared with other micro- and macroplate techniques for immune diffusion. Int Arch Allergy Appl Immunol21: 131, 1962. Address for reprints: J.-E. Brorson, M.D., Mikrobiol. lab., Guldhedsgatan 10 A , S-41346 Goteborg, Sweden Scand J Infect Dis 8
ISSN: 0036-5548 (Print) 1651-1980 (Online) Journal homepage: http://www.tandfonline.com/loi/infd19
Studies on Branhamella Catarrhalis (Neisseria Catarrhalis) with Special Reference to Maxillary Sinusitis John-Erik Brorson, Alf Axelsson & Stig E. Holm To cite this article: John-Erik Brorson, Alf Axelsson & Stig E. Holm (1976) Studies on Branhamella Catarrhalis (Neisseria Catarrhalis) with Special Reference to Maxillary Sinusitis, Scandinavian Journal of Infectious Diseases, 8:3, 151-155, DOI: 10.3109/inf.1976.8.issue-3.05 To link to this article: http://dx.doi.org/10.3109/inf.1976.8.issue-3.05
Published online: 02 Jan 2015.
Submit your article to this journal
Article views: 1
View related articles
Citing articles: 7 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=infd19 Download by: [UQ Library]
Date: 25 April 2016, At: 18:37
Scand J Infect Dis 8: 151-155. 1976
Studies on Branhamella catarrhalis (Neisseria catarrhalis) with Special Reference to Maxillary Sinusitis JOHN-ERIK BRORSON, ALF AXELSSON and STIG E. HOLM
Downloaded by [UQ Library] at 18:37 25 April 2016
From the Institute of Medical Microbiology, the Department of Clinical Bacteriology, University of Goteborg, Goteborg, the Department of Otolaryngology, Lundby Medical Center, and the Department of Clinical Bacteriology, University of Umed. Umed, Sweden
ABSTRACT. Paired sera from 97 patients with acute maxillary sinusitis were examined regarding antibodies to Branhamella catarrhalis. Precipitating antibodies were demonstrated in almost all sera both from patients and from healthy blood donors. Complement-fixing(CF) antibodies to B. catarrhalis were present in sera from 25 of the 97 patients and in one of 20 healthy blood donors. The titres were low and the titre changes when present were of a small magnitude. CF antibodies were most commonly demonstrated in the younger age groups. The patients with demonstrable CF antibodies to B. catarrhalis did not differ from patients without antibodies regardingradiological appearance or healing during therapy. Strains of B. catarrhalis were all rapidly killed by normal human serum but not in heated sera. The strains could not multiplicate significantly at an oxygen tension correspondingto about half the atmospheric value. The possible significance of the serological and bacteriological findings is discussed.
INTRODUCTION Branhamella catarrhalis, formerly known as Neisseria catarrhalis, has its natural habitat in the upper respiratory tract of man. It has been isolated from middle ear exudates of children suffering from acute middle ear suppuration (9, 15), and it has been proposed that the bacterium should be regarded as pathogenic in this condition (15). Sporadic cases of meningitis and septicemia due to this microbe have also been reported (1 1, 19). It has also been isolated, often in mixed cultures, from maxillary secretions of patients suffering from acute maxillary sinusitis (2, 10, 16). Serological studies on maxillary sinusitis. have to our knowledge not included B. catarrhalis. This fact initiated the present study of the immune response to B. catarrhalis in patients suffering from acute maxillary sinusitis. MATERIALS AND METHODS Study group The study group consists of 97 patients attending the ENT department for maxillary sinusitis. All had radiological signs of acute maxillary sinusitis with mucous membrane
thickening and secretion. The radiological method for demonstration of acute maxillary sinusitis and the evaluation of treatment has been described before (8). Sinus aspirates for bacteriological studies were obtained from all patients by puncture of the maxillary sinus after topical anesthesia with 4% lindocain. These specimens were processed as described earlier (2). Serological studies Blood was drawn by venipuncture on the first examination at the ENT department and after 15 days. Serum was recovered by centrifugation and was kept at -20°C until processed. Sera collected during the study period from 20 healthy blood donors with matched age groups served as controls. Antigens for immunodiffusion (ID) analyses were prepared from 10 different B. catarrhalis strains freshly isolated from young children with nasopharyngeal infections. The bacteria were seeded on agar plates on an antigen-free medium (13), harvested after 20 hours’ incubation at 3TC, washed 3 times in phosphate buffered saline pH 7.0. The sediment was suspended in 2 volumes of the same buffer and desintegrated in a Mickle cell desintegrator for 1 X 5 min under coolirig conditions. The preparation was centrifuged for 30 min at 2OOOOxg, and the supernate used in the ID analyses. A similar antigen was prepared from 20 different strains of Neisseria gonorrhoeae. Antigen for complement fixation (CF). Part of the bacteria harvested from the plates (see above) were washed and finally heated to 56°C for 30 min. The bacteria Scand J Infect Dis 8
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J.-E. Brorson et al.
Table I . Age and sex distribution Numbers within brackets denote number of patients within each group with demonstrable C F antibodies to B. catarrhalis Healthy blood donors (control group)
Study group Age (Y.)
Males
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c20 2 1-30 31 4 0 4 1-50 S5 I
8 (3) 9 (3) 8 (3)
2 5 (2) 32 (1 1)
Females
4 (2) (7)
19 17 17 8
(I) (3) (1)
65 (14)
Total
Males
12 (5) 28 (10) 25 (4) 19 (3) 13 (3) 97 (25)
2 2 2
employed for the C F studies were used in the highest concentration not giving anticomplementary activity. Similarly a N. gonorrhoeae suspension was prepared and analysed. ID analyses were performed by means of the microgelchamber method of Wadsworth (22). CF-analyses were made largely in accordance with Kabat and Mayer (14). The highest serum dilution giving less than 50% hemolysis was registered as the limit value. The serum dilutions started at 1 :7.5. Antisera. Rabbits were hyperimmunized by weekly intramuscular injections of 0.2 ml of the desintegrated B. catarrhalis and N. gonorrhoeae antigens respectively in complete Freund adjuvant. The immune response was followed by weekly bleedings. Reference system. To establish the antigenic pattern of the two Neisseria antigen preparations the two antigens for ID analyses were tested with their homologous rabbit hyperimmunsera in ID. Between 10 and 15 separate precipitation lines were detected in each system illustrating the multiplicity of immunogens in the antigen preparations. No cross reactions were found between the N. gonorrhoeae and B. catarrhalis precipitating antigens. Studies on B . catarrhalis Bacteria. Three strains of B. catarrhalis were used. They were all recent clinical isolates. They were identified according to Berger (4).
... .
0
0
7.5
15
30
Initial titre
Fig. 1 . Comparison between C F titres to B. catarrhalis registered initially and 15 days later in 25 persons suffering from acute maxillary sinusitis. Scand J Infect Dis 8
Females
Total
1
4 (1) 4 3 I
3 6 (1) 6 3 2
7
13 (1)
20 ( I )
1
Bacterial inoculum. In all experiments the bacterial inoculum consisted of cells grown for 5 hours in ox-broth on a rotary shaker waterbath at 37°C. Viable counts were performed by inoculating bloodagar plates with appropriate dilutions from the different systems. The plates were read after 48 hours at 37°C. Influence of serum. Blood was drawn from 2 healthy volunteers without evidence of upper respiratory tract infection the last 8 weeks. Serum was obtained by centrifugation and was kept at -20°C for a maximum period of 2 weeks before use. One portion of each serum was heated to 56°C for 30 min. The different serum samples were inoculated with cells of the 3 strains washed twice in physiologic saline. Viable counts were performed after 1.5,6 and 24 hours. Influence ofoxygen tension. The 3 strains were grown in 20 ml ox-broth in 300 ml indented flasks. Each flask was inoculated to contain about lo3 cells/ml. One flask was placed on a rotary water bath at 37°C. Three flasks of each strain were placed at 37°C in separate anaerobic jars which were evacuated to 300 mmHg. Viable counts were performed after 6, 24 and 48 hours.
RESULTS Of the 97 patients with radiological signs of acute maxillary sinusitis 25 had CF antibodies against B. catarrhalis (Table I). The titres were low and the titre changes were of a small magnitude (Fig. I). C F antibodies were most commonly demonstrated in the younger age groups. Only one serum from the control group demonstrated C F antibodies. With the exception of 6 patients, precipitating antibodies to B. catarrhalis were demonstrated in sera from the 97 patients suffering from acute maxillary sinusitis as well as in the control group. These few sera originated from persons of older age. All patients with CF antibodies had precipitating antibodies. Comparative ID analyses showed that patients and with precipitins had at least one common antibody. The
BI.anhamella catarrhalis and maxillary sinusitis /
,-f/C'
Viable count
-
lo8-
#
/
#
/
/
X
1.5
6
24lime
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Fig. 2. Influence of human serum on the growth of B. catarrhalis. -=normal human serum; - - - =human serum heated to 56°C for 30 min. Time is given in hours.
number of antibodies registered in different sera varied between I and 3. The outcome of the treatment was measured radiologically. The 25 patients with demonstrable C F antibodies to B. catarrhalis did not differ from the 72 patients without CF antibodies regarding radiological appearance of healing during identical therapy. The influence of serum on B. catarrhalis can be seen from Fig. 2. Normal human serum had a pronounced killing effect. All 3 strains investigated were killed in normal serum whereas the bacteria grew in serum heated to 56°C for 30 min. All unheated sera affected the 3 strains to a comparable degree. The oxygen tension had a marked influence on growth rate of B. catarrhalis (Fig. 3). Only slight multiplication of the bacteria was demonstrable at reduced oxygen tension. DISCUSSION There are two modes of establishing an assumed etiological association between microbes and infectious disease, i.e. demonstration of the organism and/or serological demonstration of a titre change during the course of the disease. Both methods have their disadvantages. In the case of sinusitis aspirated secretion must be obtained since the nasal flora reflects the sinus flora unreliably (2). Furth-' ermore, the transport time, the transport medium and the handling of the bacteriological samples also influence the bacteriological results. It is also possible that the microorganism inducing infection is no longer present when the sinus aspirate is analyzed. Serological examinations are more difficult to
153
perform in ordinary praxis and it is often hard to motivate the patient to return after 2-3 weeks for a second blood sample when the disease already has subsided, treated or untreated, to assure a titre change as the significant serologic diagnostic criterium. There is an obvious discrepancy between the numerous reports dealing with the bacteriology of maxillary sinusitis (La. 5, 10, 16, 17, 18, 21) and the low number of reports dealing with serology in this disease (20). Surprisingly, many of the patients (25 out of 97) had demonstrable C F antibodies to B. catarrhalis and in most of these patients a moderate change in titre was observed. The facts that C F antibodies with one exception were not observed in the control group and that titre changes were observed in many of the patients suffering from acute maxillary sinusitis suggest that the titre changes might be significant for an association with the disease. Precipitating antibodies were demonstrated in almost all sera both from patients suffering from acute maxillary sinusitis and in the control group. A possible explanation for this may be that precipitating antibodies reflect the fact that B. catarrhalis is a normal inhabitant of the upper respiratory tract and that most persons are continuously exposed to its antigens resulting in the emergence of precipitating antibodies in the same way as was earlier demonstrated for streptococcal antibodies (12). On the other hand C F antibodies are usually connected with an actual or recent infection and they are usually not demonstrable for more than some months after the disease as was clearly shown for Haemophilus influenzae (6). Bacteriological studies on maxillary sinusitis differ widely regarding the frequency with which B.
10'1
I
, 6
r
24
48 lime
Fig. 3 . Influence of oxygen tension on the growth of B. catarrhalis. -=cells grown at 760 mmHg; - - - =cells grown at 300 mmHg. Time is given in hours. Scand J Infect Dis 8
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catarrhalis has been isolated on culture (5, 10, 16, 17, 18, 21). Thus, the present serological study could indicate that the correlation between culture and serology regarding B. catarrhalis in acute maxillary sinusitis is poor. There may be many explanations to such a suggestion but it should be mentioned that a lack of correlation between cultural and serological findings exists for other bacteria too. A positive correlation between isolation of e.g. H. influenzae from the maxillary sinus and demonstration of C F antibodies to this microbe in the same patients is far from complete (20). Another explanation to the lack of correlation between isolation of B. catarrhalis and detection of C F antibodies implies the possibility that the antibody response detected against B. catarrhalis in these patients is a part of the defence mechanism against this microorganism. The present study has also shown that B . catarrhalis is markedly influenced by factors which might contribute to the fact that the microbe relatively seldom is isolated from maxillary contents of patients suffering from acute maxillary sinusitis. It is well known that B. catarrhalis does not grow under anaerobic conditions. The finding that B. catarrhalis is almost unable to multiplicate at an oxygen tension corresponding to about half the atmospheric value might be of some importance in explaining the fact that B. catarrhalis is seldom isolated from maxillary aspirates. It is e.g. known that the oxygen content in a maxillary sinus with impaired drainage rapidly decreases to a value corresponding to about half the atmospheric value (1). It is also a well known fact that ostial dysfunction is very common in acute maxillary sinusitis making low oxygen tension quite plausible. It was also found that normal serum rapidly killed strains of B. catarrhalis and that this killing effect of serum was abolished by heat inactivation. This could indicate that the killing might be due to a complement-demanding process. No attempt was made to elucidate the factors responsible for the killing, but this study has also shown that it is very common to find precipitating antibodies to B. catarrhalis in sera from healthy persons. Many bacterial species have been demonstrated in acute maxillary sinusitis. Some degree of correlation has been observed between infecting bacterial species and initial radiological appearance and healing during therapy (3). In this study it was found that neither the initial radiological appearance nor Scotid J Infect Dis 8
the healing process during therapy could be correlated to the presence of C F antibodies to B. catarrhalis. The demonstration of C F antibodies to a microbe often present in the vicinity of the maxillary sinus is no proof that the microbe in fact is incriminated in acute maxillary sinusitis. In a parallel study, antibodies to other bacteria generally thought to be of etiological significance in acute maxillary sinusitis were often demonstrated alone or in combination with C F antibodies to B . catarrhalis (7). However, in some cases with negative culture for bacteria the only positive serological finding was the demonstration of C F antibodies to B. catarrhalis. It remains to be elucidated whether this indicates that B . catarrhalis should be regarded as being of etiological significance in acute maxillary sinusitis. Antigenic analyses of B . catarrhalis have clearly indicated that this microorganism should not be included among the Neisseria species. Rather it belongs to the Moraxella group. It should be noted that the Moraxella group is not antigenically related to H. influenzae or pneumococci which are generally incriminated in acute maxillary sinusitis. This indicates that the observed C F antibodies probably should be regarded as specific and not due to cross reactions with the bacteria most commonly seen in maxillary sinusitis. ACKNOWLEDGEMENT The excellent technical assistance of Mrs Ingrid Wargskog is gratefully acknowledged.
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maxillary sinus. Int Rhino1 9: 69, 1971. 2. Axelsson, A. & Brorson, J.-E.: The correlation between bacteriological findings in the nose and maxillary sinus in acute maxillary sinusitis. Laryngoscope 83: 2003, 1973. 3. Axelsson, A. & Chidekel, N.: Symptomatology and bacteriology correlated to radiology in acute maxillary sinusitis. Acta Otolaryngol (Stockh) 74: 118, 1972. 4. Berger, U.: Die anspruchlosen Neisserien. Ergeb Mikrobiol36: 97, 1963. 5 . Bjorkwall, T.: Bacteriological examinations in maxillary sinusitis. Acta Otolaryngol (Stockh), Suppl. 83: 1, 1950. 6. Branefors-Helander, P.: Serological studies of Haemophilus influenzae with special reference to otitis media infections. Dissertation, University of Goteborg, Sweden 1973.
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