Acta Oto-Laryngologica. 2014; 134: 1022–1028
ORIGINAL ARTICLE
Acta Otolaryngol Downloaded from informahealthcare.com by University of Melbourne on 09/15/14 For personal use only.
Immunologic findings in young children with early onset of acute otitis media
MARIE GISSELSSON-SOLÉN 1, ANN HERMANSSON1, ÅSA MELHUS2 & NICHOLAS BRODSZKI3 1
Department of Otorhinolaryngology, Head and Neck Surgery, Lund University Hospital, Lund, 2Department of Medical Sciences/Section of Clinical Bacteriology, Uppsala University, Uppsala and 3Department of Pediatrics, Lund University Hospital, Lund, Sweden
Abstract Conclusion: No significant differences in the number of immune aberrations were seen between children with or without severe recurrent acute otitis media (rAOM); however, subnormal values of immunological markers were found more often than expected, and 4 of the 60 children had treatment-requiring immune deficiencies. Objective: Minor immunologic aberrations have been reported to be more frequent in children with rAOM. Immune investigation is recommended in children with severe rAOM, defined as six or more AOM episodes per year. The purpose of this study was to describe immunological findings in young children at high risk of developing rAOM, and to relate these to the number of expected aberrations and to the presence of severe rAOM. Methods: A total of 109 children at risk of developing rAOM were offered immune investigation including complement function, immunoglobulins with subclasses and cellular immunity. Results: Sixty patients were tested, 31 of whom had severe rAOM and 12 of whom did not develop rAOM. Low levels of IgG2 (27%), C1q (31%) and mannan-binding lectin (21%) were found up to eight times as often as expected. Although subnormal values were more frequent among children with severe rAOM, the study was too small to provide reliable evidence of any difference. Four children were diagnosed with immune deficiencies that required treatment.
Keywords: Recurrent acute otitis media, rAOM, AOM, IgG2, MBL, C1q, IgA, vaccination
Introduction Acute otitis media (AOM) is the most common bacterial disease in childhood, and about 10% of all children suffer from recurrent AOM (rAOM), defined as at least three episodes in 6 months or four episodes in a year [1]. This condition usually resolves after 2 years of age [1]. The aetiology of rAOM is not entirely clear. It is generally considered that children with rAOM do not suffer from severe immune deficiencies [2], but minor immunological aberrations have been observed. One of these is low levels of the IgG subclass IgG2, something that usually resolves spontaneously after a few years [3–5]. An association between rAOM and low levels of the
complement protein C1q has also been pointed out [6]. In recent years, a connection between deficiency of mannose-binding lectin (MBL) – a complement protein vital for the activation of the lectin pathway of the complement system – and recurrent upper airway infections has been shown [7]. In Sweden, it has been suggested that children with severe rAOM, defined as at least six AOM episodes in a year, should be subject to immune investigation [8]. The purpose of this study was to investigate immunological aberrations in young children at high risk of developing rAOM and to relate the results to what would have been expected in the general population as well as to the presence of severe rAOM.
Correspondence: Marie Gisselsson-Solén MD PhD MSc, Department of Otorhinolaryngology, Head & Neck Surgery, 221 85 Lund, Sweden. Tel: +44 756 427 9917. Fax: +46 46 171758. E-mail: [email protected] or E-mail: [email protected]
(Received 20 January 2014; accepted 27 February 2014) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2014.902539
Acta Otolaryngol Downloaded from informahealthcare.com by University of Melbourne on 09/15/14 For personal use only.
Immunologic findings and recurrent acute otitis media
1023
Material and methods
Immunological investigation
Study design and patients
Blood samples were collected in SST and EDTA tubes. Serum and plasma were kept refrigerated until analysis at the Department of Clinical Immunology and Transfusion Medicine, Regional and University Laboratories at Lund University Hospital. The following parameters were measured: complement, immunoglobulins and cellular immunity, as described below. Complement: C3, C4, qualitative function of the classical and alternative pathways, properdin, C1q, quantative function of the lectin pathway; and MBL. Immunoglobulins: IgG, IgA, IgM and IgG subclasses. Cellular immunity: CD3 (T cells), CD4 (helper T cells), CD8 (cytotoxic T cells), CD19 (B cells), CD4/CD8 ratio, CD16 + CD56 (natural killer cells) and total lymphocyte concentration. Qualitative complement function (classical and alternative pathways) was measured by haemolysis in gel as described previously [10] and quantitative complement function (lectin pathway) by a commercially available ELISA method (Wieslab COMPL MP320, Euro Diagnostica, Malmö, Sweden) [10,11]. Complement proteins were measured by rocket immunoelectrophoresis [12] and nefelometry. MBL was measured by a capture-ELISA technique using commercially available monoclonal antibodies [13]. Immunoglobulins with subclasses, C3 and C4 were measured quantitatively by nefelometry using an Immage 800 Nefelometer (Beckman Coulter, Brea, California, USA) or turbidometry using an ABX Pentra 400 (Horiba Medical, Montpelier, France). A supplementary analysis of immunoglobulins in the form of agarose electrophoresis was also performed in each case to detect M-components and as an aid to interpretation of the results. Lymphocyte markers were measured quantitatively by flow cytometry FC 500, Beckman Coulter.
This study was a secondary outcome in a randomized, single-blinded vaccination trial, investigating the effect of heptavalent pneumococcal conjugate vaccine in children at high risk of developing rAOM. The trial was approved by the Ethics Committee at Lund University Hospital. Patients were included between March 2003 and June 2007. For inclusion, the child had to have had at least one AOM episode confirmed by an otorhinolaryngologist before the age of 6 months, thus estimating that 80% of the children would develop rAOM [9]. Half of the children were randomized to receive vaccination with heptavalent conjugate pneumococcal vaccine (Prevenar, Wyeth-Lederle, now Pfizer); however, the use of a control vaccine, such as a hepatitis B vaccine, was not approved by the Ethics Committee. Exclusion criteria were allergy to the vaccine, anatomical or chromosomal abnormalities, previously diagnosed immune deficiencies, prematurity, prior administration of gammaglobulin or pneumococcal vaccine and a history of idiopathic thrombocytopenic purpura. The children were followed closely for 3 years: during the first year after inclusion, all children were examined by one of the two study doctors (both otorhinolaryngologists) at regularly scheduled visits every other month. In addition, during the entire 3-year follow-up, the children were examined whenever parents suspected a new episode of AOM. A scheduled visit to one of the study doctors was carried out 3 years after inclusion. Otomicroscopy was performed at all doctors’ visits. All study children were offered immunological testing in the form of analyses of complement function, immunoglobulin levels and distribution of lymphocyte subpopulations in peripheral blood, see below.
Statistical analyses Diagnosis AOM was defined as a bulging eardrum and opaque fluid in the middle ear in a child with symptoms of an acute infection. A new episode of AOM was diagnosed if the child, after completing treatment with antibiotics and having a period with no clinical signs of AOM, had new symptoms and otomicroscopic findings as described above. Recurrent AOM was defined as at least three episodes during a 6-month period or four episodes in a year. Severe rAOM was defined as at least six AOM episodes in 1 year.
Measurements were classified as normal or subnormal according to the age-related reference values. The number of subnormal values was then calculated for the group as a whole, but was also correlated to the presence of severe rAOM. The statistical package Stata 13.0 was used for all statistical analyses. Group differences in binary variables were examined with the c2 test, and relative risks were examined with logistic regression, controlling for age and gender. So as not to have to assume a linear relationship between age and a certain risk factor, age was treated as a categorical variable. According to the
1024
M. Gisselsson-Solén et al.
literature, the age of 2 years is an important cut-off point concerning the influence of immunological aberrations on recurrent infections. To achieve groups of similar and adequate size, the children were therefore divided into 2-year age groups.
Acta Otolaryngol Downloaded from informahealthcare.com by University of Melbourne on 09/15/14 For personal use only.
Results A total of 109 children were included in the trial, 105 of which completed the 3-year follow-up. Mean age at inclusion was 5.0 months, and 70 (64%) of the children were male. Sixty parents accepted immune investigation, 80% of these patients had rAOM, with eight AOM episodes during the 3-year follow-up as a median. The corresponding figures for those who declined testing were 84% and seven episodes. Figure 1 illustrates the study flow chart. Blood tests for immune investigation were voluntary, causing the age of the children at testing to vary considerably. The mean age at immunological investigation was 32 months, and 28 of the patients were under 2 years at the time of testing. Of the 60 children who underwent immunological investigation, 31 belonged to the control group and 29 to the vaccine group. Four children were diagnosed with immune deficiencies that were considered to require treatment (three received gammaglobulins and one patient extra vaccinations) and in one patient, a rare chromosomal abnormality (deletion of chromosome 13) was discovered. Thirty-one of the 60 patients had severe rAOM as defined above, whereas 12 did not develop rAOM at all. The number of subnormal results for each parameter is shown together with the corresponding expected number in Table I [14–16]. The most
commonly found aberrations were IgG2 (27%), C1q (31%) and MBL (21%), although low levels of IgA were found in 12% of the patients, low IgG4 in 13% and low lymphocyte levels in 14%. Table II shows the relation between the most prevalent aberrations and severe rAOM. After adjusting for age and gender, the parameter associated with the largest increase in the odds ratio for severe rAOM was IgG2, which conferred an almost threefold increase in the odds of severe rAOM; however, the study was too small to provide reliable evidence of this effect being true. Apart from low lymphocyte levels, measurements below the age-specific reference values of cellular immunity parameters were found rarely. There was some evidence of aberrations being more frequent among children below 2 years of age, especially for C1q (Table III). Co-variations between the different parameters were examined, and there was good evidence for a correlation between subnormal levels of IgG2 and C1q (p = 0.014). No correlation was seen between other parameters. The four patients with immune deficiencies requiring treatment, and the patient with chromosome 13 deletion, all fulfilling the definition of severe rAOM, showed multiple aberrations (Table IV). Discussion In this small descriptive study performed on mainly otitis-prone children, some aberrations of the immune system were found more than eight times as often as would be expected. The most common aberrations were low IgG2, low C1q and MBL deficiency, being found more often among children below 2 years of age.
109 included
4 drop-outs
105 offered immune investigation
45 declined immune investigation
60 immune investigation
31 severe rAOM (14 vaccine group, 17 control group) 4 immune deficiencies, 1 chromosomal aberration
29 without severe rAOM (15 vaccine group, 14 control group)
Figure 1. Patient flow chart.
Immunologic findings and recurrent acute otitis media
1025
Table I. Aberrant immunological findings. Reference value
Acta Otolaryngol Downloaded from informahealthcare.com by University of Melbourne on 09/15/14 For personal use only.
Characteristic
Expected no. of aberrant results*
No. of aberrant results/no. of tested children (%)†
No. with severe rAOM
No. of AOM episodes‡, median (95% CI)
Low C3
0.77–1.38
ORIGINAL ARTICLE
Acta Otolaryngol Downloaded from informahealthcare.com by University of Melbourne on 09/15/14 For personal use only.
Immunologic findings in young children with early onset of acute otitis media
MARIE GISSELSSON-SOLÉN 1, ANN HERMANSSON1, ÅSA MELHUS2 & NICHOLAS BRODSZKI3 1
Department of Otorhinolaryngology, Head and Neck Surgery, Lund University Hospital, Lund, 2Department of Medical Sciences/Section of Clinical Bacteriology, Uppsala University, Uppsala and 3Department of Pediatrics, Lund University Hospital, Lund, Sweden
Abstract Conclusion: No significant differences in the number of immune aberrations were seen between children with or without severe recurrent acute otitis media (rAOM); however, subnormal values of immunological markers were found more often than expected, and 4 of the 60 children had treatment-requiring immune deficiencies. Objective: Minor immunologic aberrations have been reported to be more frequent in children with rAOM. Immune investigation is recommended in children with severe rAOM, defined as six or more AOM episodes per year. The purpose of this study was to describe immunological findings in young children at high risk of developing rAOM, and to relate these to the number of expected aberrations and to the presence of severe rAOM. Methods: A total of 109 children at risk of developing rAOM were offered immune investigation including complement function, immunoglobulins with subclasses and cellular immunity. Results: Sixty patients were tested, 31 of whom had severe rAOM and 12 of whom did not develop rAOM. Low levels of IgG2 (27%), C1q (31%) and mannan-binding lectin (21%) were found up to eight times as often as expected. Although subnormal values were more frequent among children with severe rAOM, the study was too small to provide reliable evidence of any difference. Four children were diagnosed with immune deficiencies that required treatment.
Keywords: Recurrent acute otitis media, rAOM, AOM, IgG2, MBL, C1q, IgA, vaccination
Introduction Acute otitis media (AOM) is the most common bacterial disease in childhood, and about 10% of all children suffer from recurrent AOM (rAOM), defined as at least three episodes in 6 months or four episodes in a year [1]. This condition usually resolves after 2 years of age [1]. The aetiology of rAOM is not entirely clear. It is generally considered that children with rAOM do not suffer from severe immune deficiencies [2], but minor immunological aberrations have been observed. One of these is low levels of the IgG subclass IgG2, something that usually resolves spontaneously after a few years [3–5]. An association between rAOM and low levels of the
complement protein C1q has also been pointed out [6]. In recent years, a connection between deficiency of mannose-binding lectin (MBL) – a complement protein vital for the activation of the lectin pathway of the complement system – and recurrent upper airway infections has been shown [7]. In Sweden, it has been suggested that children with severe rAOM, defined as at least six AOM episodes in a year, should be subject to immune investigation [8]. The purpose of this study was to investigate immunological aberrations in young children at high risk of developing rAOM and to relate the results to what would have been expected in the general population as well as to the presence of severe rAOM.
Correspondence: Marie Gisselsson-Solén MD PhD MSc, Department of Otorhinolaryngology, Head & Neck Surgery, 221 85 Lund, Sweden. Tel: +44 756 427 9917. Fax: +46 46 171758. E-mail: [email protected] or E-mail: [email protected]
(Received 20 January 2014; accepted 27 February 2014) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2014.902539
Acta Otolaryngol Downloaded from informahealthcare.com by University of Melbourne on 09/15/14 For personal use only.
Immunologic findings and recurrent acute otitis media
1023
Material and methods
Immunological investigation
Study design and patients
Blood samples were collected in SST and EDTA tubes. Serum and plasma were kept refrigerated until analysis at the Department of Clinical Immunology and Transfusion Medicine, Regional and University Laboratories at Lund University Hospital. The following parameters were measured: complement, immunoglobulins and cellular immunity, as described below. Complement: C3, C4, qualitative function of the classical and alternative pathways, properdin, C1q, quantative function of the lectin pathway; and MBL. Immunoglobulins: IgG, IgA, IgM and IgG subclasses. Cellular immunity: CD3 (T cells), CD4 (helper T cells), CD8 (cytotoxic T cells), CD19 (B cells), CD4/CD8 ratio, CD16 + CD56 (natural killer cells) and total lymphocyte concentration. Qualitative complement function (classical and alternative pathways) was measured by haemolysis in gel as described previously [10] and quantitative complement function (lectin pathway) by a commercially available ELISA method (Wieslab COMPL MP320, Euro Diagnostica, Malmö, Sweden) [10,11]. Complement proteins were measured by rocket immunoelectrophoresis [12] and nefelometry. MBL was measured by a capture-ELISA technique using commercially available monoclonal antibodies [13]. Immunoglobulins with subclasses, C3 and C4 were measured quantitatively by nefelometry using an Immage 800 Nefelometer (Beckman Coulter, Brea, California, USA) or turbidometry using an ABX Pentra 400 (Horiba Medical, Montpelier, France). A supplementary analysis of immunoglobulins in the form of agarose electrophoresis was also performed in each case to detect M-components and as an aid to interpretation of the results. Lymphocyte markers were measured quantitatively by flow cytometry FC 500, Beckman Coulter.
This study was a secondary outcome in a randomized, single-blinded vaccination trial, investigating the effect of heptavalent pneumococcal conjugate vaccine in children at high risk of developing rAOM. The trial was approved by the Ethics Committee at Lund University Hospital. Patients were included between March 2003 and June 2007. For inclusion, the child had to have had at least one AOM episode confirmed by an otorhinolaryngologist before the age of 6 months, thus estimating that 80% of the children would develop rAOM [9]. Half of the children were randomized to receive vaccination with heptavalent conjugate pneumococcal vaccine (Prevenar, Wyeth-Lederle, now Pfizer); however, the use of a control vaccine, such as a hepatitis B vaccine, was not approved by the Ethics Committee. Exclusion criteria were allergy to the vaccine, anatomical or chromosomal abnormalities, previously diagnosed immune deficiencies, prematurity, prior administration of gammaglobulin or pneumococcal vaccine and a history of idiopathic thrombocytopenic purpura. The children were followed closely for 3 years: during the first year after inclusion, all children were examined by one of the two study doctors (both otorhinolaryngologists) at regularly scheduled visits every other month. In addition, during the entire 3-year follow-up, the children were examined whenever parents suspected a new episode of AOM. A scheduled visit to one of the study doctors was carried out 3 years after inclusion. Otomicroscopy was performed at all doctors’ visits. All study children were offered immunological testing in the form of analyses of complement function, immunoglobulin levels and distribution of lymphocyte subpopulations in peripheral blood, see below.
Statistical analyses Diagnosis AOM was defined as a bulging eardrum and opaque fluid in the middle ear in a child with symptoms of an acute infection. A new episode of AOM was diagnosed if the child, after completing treatment with antibiotics and having a period with no clinical signs of AOM, had new symptoms and otomicroscopic findings as described above. Recurrent AOM was defined as at least three episodes during a 6-month period or four episodes in a year. Severe rAOM was defined as at least six AOM episodes in 1 year.
Measurements were classified as normal or subnormal according to the age-related reference values. The number of subnormal values was then calculated for the group as a whole, but was also correlated to the presence of severe rAOM. The statistical package Stata 13.0 was used for all statistical analyses. Group differences in binary variables were examined with the c2 test, and relative risks were examined with logistic regression, controlling for age and gender. So as not to have to assume a linear relationship between age and a certain risk factor, age was treated as a categorical variable. According to the
1024
M. Gisselsson-Solén et al.
literature, the age of 2 years is an important cut-off point concerning the influence of immunological aberrations on recurrent infections. To achieve groups of similar and adequate size, the children were therefore divided into 2-year age groups.
Acta Otolaryngol Downloaded from informahealthcare.com by University of Melbourne on 09/15/14 For personal use only.
Results A total of 109 children were included in the trial, 105 of which completed the 3-year follow-up. Mean age at inclusion was 5.0 months, and 70 (64%) of the children were male. Sixty parents accepted immune investigation, 80% of these patients had rAOM, with eight AOM episodes during the 3-year follow-up as a median. The corresponding figures for those who declined testing were 84% and seven episodes. Figure 1 illustrates the study flow chart. Blood tests for immune investigation were voluntary, causing the age of the children at testing to vary considerably. The mean age at immunological investigation was 32 months, and 28 of the patients were under 2 years at the time of testing. Of the 60 children who underwent immunological investigation, 31 belonged to the control group and 29 to the vaccine group. Four children were diagnosed with immune deficiencies that were considered to require treatment (three received gammaglobulins and one patient extra vaccinations) and in one patient, a rare chromosomal abnormality (deletion of chromosome 13) was discovered. Thirty-one of the 60 patients had severe rAOM as defined above, whereas 12 did not develop rAOM at all. The number of subnormal results for each parameter is shown together with the corresponding expected number in Table I [14–16]. The most
commonly found aberrations were IgG2 (27%), C1q (31%) and MBL (21%), although low levels of IgA were found in 12% of the patients, low IgG4 in 13% and low lymphocyte levels in 14%. Table II shows the relation between the most prevalent aberrations and severe rAOM. After adjusting for age and gender, the parameter associated with the largest increase in the odds ratio for severe rAOM was IgG2, which conferred an almost threefold increase in the odds of severe rAOM; however, the study was too small to provide reliable evidence of this effect being true. Apart from low lymphocyte levels, measurements below the age-specific reference values of cellular immunity parameters were found rarely. There was some evidence of aberrations being more frequent among children below 2 years of age, especially for C1q (Table III). Co-variations between the different parameters were examined, and there was good evidence for a correlation between subnormal levels of IgG2 and C1q (p = 0.014). No correlation was seen between other parameters. The four patients with immune deficiencies requiring treatment, and the patient with chromosome 13 deletion, all fulfilling the definition of severe rAOM, showed multiple aberrations (Table IV). Discussion In this small descriptive study performed on mainly otitis-prone children, some aberrations of the immune system were found more than eight times as often as would be expected. The most common aberrations were low IgG2, low C1q and MBL deficiency, being found more often among children below 2 years of age.
109 included
4 drop-outs
105 offered immune investigation
45 declined immune investigation
60 immune investigation
31 severe rAOM (14 vaccine group, 17 control group) 4 immune deficiencies, 1 chromosomal aberration
29 without severe rAOM (15 vaccine group, 14 control group)
Figure 1. Patient flow chart.
Immunologic findings and recurrent acute otitis media
1025
Table I. Aberrant immunological findings. Reference value
Acta Otolaryngol Downloaded from informahealthcare.com by University of Melbourne on 09/15/14 For personal use only.
Characteristic
Expected no. of aberrant results*
No. of aberrant results/no. of tested children (%)†
No. with severe rAOM
No. of AOM episodes‡, median (95% CI)
Low C3
0.77–1.38
