Clin. Otolaryngol. 1992, 17, 195-199

Occurrence of Streptococcus pneumoniae and Haemophilus influenzae in otitis media with effusion LARS-ERIC STENFORS & SIMO RAISANEN* Departments of Otolaryngology, University of Tromso, Tromso, Norway and Central Hospital of Keski-Pohjanmaa, Kokkola, Finland, and the *Clinical Laboratory, Central Hospital of Keski-Pohjanmaa. Kokkola. Finland Accepted for publication 18 June 1991 STENFORS L . - E . & R A N A N E N

s.

(1992) Clin. Otolaryngol. 17, 195-199

Occurrence of Streptococcus pneumoniae and Haemophilus infruenzae in otitis media with effusion Eighty mucoid effusion samples obtained from 56 patients with otitis media with effusion (OME) were subjected to quantitative and qualitative bacteriological analysis using standard culturing methods, direct microscopy and immunofluorescent assay. 30% of the samples contained culture-positive pathogens ( H . infuenzae, S . pneumoniae, B. catarrhalis), with counts never exceeding 5 x lo5 per ml. In addition, 19% of the samples had dormant H . infuenzae and S . pneurnoniae, which did not grow on standard agar plates. Viable and dormant bacteria, as well as bacterial remnants, play a crucial role in the pathogenesis of O M E and similarities between OME and reactive arthritis, i.e. Lyme arthritis, Reiter’s syndrome and rheumatic fever, are evident. Keywords secretory otitis media reactive arthritis bacterial quantity dormant bacteria

Otitis media with effusion (syn. secretory otitis media, OME) refers to the presence of a middle ear effusion behind an intact tympanic membrane but without the acute signs or symptoms of infection.’ Whether O M E has an infectious origin, i.e. bacteria in the effusion material, has been a very controversial question. In earlier reports the fluid has been considered sterile, whereas more recent studies have reported positive bacterial cultures 20-50% of the time.24 The microorganisms cited are Streptococcus pneumoniae, Haemophilus infuenzae, Branhamella catarrhalis, Group A streptococci, Staphylococcus aureus and coagulase-negative staphylococci.2-6 It must be mentioned that both S. aureus and coagulase-negative staphylococci belong to the normal commensal flora of the external auditory canal and may cause false-positive results when aspirating material from the middle ear cleft. In a previous study’ we presented a method of routine quantification of bacteria in middle ear effusions. We found that middle ear pathogens could be present in numbers up to 5 x 10S/mlin mucoid effusions. By contrast, bacterial counts Correspondence: Dr L.-EStenfors, Department of Otolaryngology, Central Hospital of Keski-Pohjanmaa, SF-67200 Kokkola, Finland.

acridine orange

middle ear pathogens

reached roughly 107/ml in mucopurulent effusions and 199/ml in purulent effusions, respectively,x simultaneously with a complete alteration in the clinical picture of the disease. The present study was undertaken in order to evaluate more closely the content of H. infuenzae and S. pneumoniae bacteria in mucoid effusions. Particular attention was paid to identification of dormant S . pneumoniae and H . infuenzae, not detectable by traditional culture techniques.

Material and methods Fifty-six children, of whom 30 were boys, and representing 80 ears affected by OME, comprised the study material. The age range of the patients was 1-12 years (mean 3.8 years). No antibiotics had been administered within 2 weeks of the examination. All samples were taken at the time of insertion of a tympanostomy tube and/or adenoidectomy under general anaesthesia. Before taking samples from the middle ear, the external auditory meatus was cleaned and washed twice with a 70% alcoholic solution. Using a microscope, a myringotomy was performed in the anterior/inferior quadrant of the drum. Through the perforation a sterile cannula, connected to a

195

196 L.-E.Stenf0r.y and S.Raisanen

syringe, was inserted into the middle ear cavity, care being taken to avoid touching the canal walls. Effusion material was then aspirated into the sterile syringe. The effusion material was in every case extremely viscous, thick and gluelike. The samples were immediately transferred to the clinical laboratory for processing. The effusion material was homogenized by repeated squeezing through successively finer cannulas, after which it was again aspirated into the syringe and investigated in the following ways ( I ) Standard culturing on blood agar and chocolate agar plates (2) Microscopy after staining with acridine orange (3) Antibody-bascd identification of dormant H . influenme and S. pneumoniue using an immunofluorescent assay. For the immunolluorescent studies the following antisera and methods were used: S. pneurnoniae (direct FA technique using rabbit polyvalent 7. pneumonk fluorescein-conjugated antiserum, and I / . igfluenzae (indirect FA technique using rabbit polyvalent H . ir~fiuenzaeantiserum, and species specific fluorescein-labelled goat antirabbit serum, respectively.

Table 1. Laboratory findings in the culture-positive (S. pneumoniue, H. in/fuen;ne or B. cuturrhulis present) middle ear effusions collected from O M E patients. Culture-positive sample at direct microscopy means that the bacterial denoted by quantity was less than 104/rnl Acridine orange stain Bact/rnl efTusion

No. ..

~~

.

.

~~

I 2 3 4

5 6

I

2 x 10'

8

-

9 10

II 12 13 14

3 x 10' 6 x 10'' 5 x 104 -

I5 16

2 x 10' 4 x to5 I x 105

17 18

I

19 20 21

22 23 24

105

6 x 10' -

3 x 10' 2 x 104 4 x 104

Culture results Pathogenic bacteria ~

~

Results Table 1 shows that 30% (24/80) of the mucoid effusions harboured culture-positive bacteria (Figure 1). H . influenzue was the predominant bacterial species (42%), followed by B. catarrhalis (33%) and S . pnewmoniae (25%). Standard culturing was slightly more sensitive than acridine staining, as the latter method required a bacterial content of lo4 per ml for detection. Table 2 shows that 19% (15/80) of the samples were culture-negative harbouring dormant S.pneumoniue o r H . influenzue bacteria (Figure 2). The predominant microorganism was I I . influenzae which was found in 1 1 out of 15 samples. In one case both dormant S. pneumoniae and H . injluenzae were observed simultaneously. No Group A streptococci, coagulase-negative staphylococci or S . aureus were found in the samples examined. Neutrophils were the predominant leucocytes found in the effusions. Lymphocytes and macrophages were also observed in the acridine orange stained material. Only in 4% (3/80) of the samples were no inflammatory cells found. These samples were culture-negative and no dormant bacteria could be identified using iinmunofluoresccnt assay.

Discussion In the present study middle car pathogens occurred in 30% of the effusions. However, the number of these microorganisms was not high and did not exceed 5 x los per ml (Figure I). In an additional 19% of the cases dormant H . injfuenzae and S . pncwnoniuc* were present. We did not

~~~~~~

5.curarrha1i.v S. pneumoniar S.pneumoniae S. pneumoniae H. influeniue H . inpuenzae B. ratarrhalis B. cururrhalis S. pneumoniae S. pneumoniae H. influenzae H. inpuenzae B. catarrhulis H . influenzae H. influenzae H. influenzue B. catarrhalis S . pneumoniae H. influenme B. catarrhah B. cararrhalis B. catarrhulis H . influenme H . influen:ae

Table 2. Laboratory findings in culture-negative middle ear elTusions, positive when treated with antiserum against S. pneumoniae and/or H. inJluenzae Immunotluorescence findings

No.

H. influen:ae

S. pneuriionrae ~-

1 2 3 4 5

6

I 8 9 10 I1 12 13 14 15

t

+

+ + +

-

+ + 4+ + + + 4-

+ + +

Bacteria and otitis media with eflision

Figure 1 . Photograph of mucoid efrusion showing H . in//uenzue (arrows), polyniorphonuclear leucocytes (P). lymphocyte (L) and a niacrophage (M). Bacterial count 2 x lo4 per nil. Acridine orange stain. x 1750.

Figure 2. Photograph of culture-negative mucoid effusion showing dormant H. influenzue (arrows). Indirect FA technique using rabbit polyvalent H . injhenzae antiserum and species specific fluorescein-labelled goat antirabbit serum. x 2300.

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198 L.-EStenjors and S.Raisanen

search for dormant B. catarrhalis. Against this background it seems natural to suggest that OME results from an incomplete resolution of acute purulent otitis media and/or mucopurulent otitis media with a bacterial content of the magnitude 107-10L(/m1.* When bacteria are introduced into an otherwise sterile middle ear cavity, a number of protective mechanisms come into play including chemical and mechanical protection, as well as specific and non-specific humoral and cellular elements. Polymorphonuclear leucocytes, shown in the present study in abundance, are important for phagocytosis of the bacteria.’.’’ In addition, the presence of lymphocytes and macrophages showed that both local and systemic immune responses occurred in the middle ear mucosa during OM E. Furthermore, the acidic glycosaminoglycans, so important for giving the mucoid effusion material its viscous properties, have antibacterial properties.”.” ‘The mucus binds bacteria and thus prevents them from attaching to mucosal cells where they can colonize.13 The present study shows that in OME the antibacterial system is sufficiently powerful to prevent active infection by bacteria in the majority of cases. This agrees with findings in an earlier in-vitro studyi4 where S. pneumoniue and H . injuenzae were injected into mucoid effusions. It was shown that these bacteria survived for only a couple of days in this environment. However, the simultaneous presence of viable or non-viable B. cutarrhalis could markedly prolong the survival potential of both S. pneumoniue and H. i ~ ~ f i u e n z a e However, .~~ it is possible that bacterial products in the middle ear effusion could cause or sustain the inflammation in OME. Middle ear pathogens contain biologically potent endotoxins (lipopolysaccharides) in the membrane complex, which mediate an inflammatory response.’(’ Furthermore, bacterial components form immune complexes, which certainly play a part in OME.” The fact that a high concentration of rheumatoid factor (anti-immunoglobulin antibodies) has been found in 80% of effusions” leads us to draw parallels between the effusion production in OME and that found in various forms of reactive arthritis, e.g. Lyme arthritis, Reiter’s syndrome, rheumatic These reactive conditions may result from prior localization of bacterial remnants o r dormant, fastidious bacteria in the affected joint where they act as persisting antigens. Classic culture techniques would not detect the presence of these microbial antigens. If the same phenomenon is responsible also in OME, then the infectious focus must lie close to the middle ear cleft, i.e. in the nasopharynx,6,2’where middle ear pathogens are found even in individuals with normal ears. In the final stage of OME, bacteria and inflammatory cells are not involved as the effusion is devoid of these cellular elements. The effusion merely forms an inert mass which is highly viscous and extremely hydrophilic. The antigenic and irritant properties of this material are certainly minimal and

clinical symptoms are a result only of interference with the ossicular chain and acoustic apparatus of the middle ear. This process is in most cases irreversible, as the middle ear cavity has n o method of removal of this type of material.

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factor in otitis media with effusion. Arch. Otoluryngol. 110, 20 279-280 K., JALKANEN S . , VON ESSENR.,LAHESMAA-RANTALA 21 19 GRANFORS R.,ISOMAKI o.,PEKKOLA-HEINO K.,MERILAHTI-PALO R.,SAARIO R., ISOMAKI H. & TOIVANEN A. (1989) Yersinia antigens in synovial-fluid cells from patients with reactive arthritis. N . Eng. J . Med. 320, 2 16-22 I

FOXA. (1990) Role of bacterial debris in inflammatory diseases of the joint and eye. APMIS 98, 957-968 STENFORS L.-E. & %ISANEN S. (1990) Occurrence of middle ear pathogens in the nasopharynx o f young individuals. A quantitative study in four age groups. Acta Otolaryngol. (Stockh.) 109, 142-148