Otitis media is a disease that affects many children around the world. Streptococcus pneumoniae represents the prevalent causative organism and nasopharyngeal colonization is a requisite antecedent for the development of otitis media.1,2 Yet rates of colonization per se may not correlate with middle ear disease.
Australian indigenous children have 1 of the highest rates of otitis media worldwide and disease typically occurs very early in life. In some Australian indigenous communities, 50% of infants had otitis media with effusion by the age of 30 days and 60% had suffered a perforation by 12 months.3 Approximately two-thirds of all Australian indigenous children entering primary school have a conductive hearing loss resulting from chronic otitis media.4–6
Australian indigenous infants become colonized with S. pneumoniae immediately after birth and by 3 months of age, >80% of infants are colonized.7 Remarkably, other populations such as Gambian infants also experience high rates of colonization with S pneumoniae in early infancy, yet do not experience high rates of otitis media. A cross-sectional survey, before the introduction of national pneumococcal conjugate vaccine, found that between 75% and 80% of healthy Gambian infants were colonized with pathogenic pneumococci by 3 months of age. However, rates of severe ear disease in this population remain low.8
In 1994, the World Health Organization examined the worldwide disease burden of chronic suppurative otitis media. The burden was divided into 4 prevalence categories. Gambian infants were in the lowest prevalence category (<1%), while Australian indigenous infants were in the highest category (>4%). Australian indigenous infants also had the highest prevalence of tympanic membrane perforation (28–43%) among all populations surveyed. The World Health Organization concluded that Australian indigenous infants constitute a special high risk group.9
The reasons why rates of severe ear disease are considerably higher in Australian indigenous children than most other disadvantaged populations including Gambian infants whose socioeconomic conditions are considered similar10 and who experience similar heavy nasopharyngeal colonization from early life are poorly understood.
Antibody responses to bacterial polysaccharides are considered to be an important component of the immune defense against encapsulated bacteria such as pneumococci. As humoral immune responses are poorly developed in the neonatal period,11 protection against otitis media in early life relies predominantly on maternally derived IgG antibodies acquired through placental transfer and secretory IgA in breast milk.12 This transfer is an active process mediated by specific receptors on the placental syncytiotrophoblast. The efficiency of placental transfer varies for the different subclasses of IgG, with IgG1 detected at higher concentrations in cord blood than in maternal blood and IgG2 lower in cord than maternal blood. IgG2 is important in protection against encapsulated bacteria like the pneumococcus. Factors that influence the placental transfer of IgG include low birth weight, prematurity, maternal immunoglobulin levels, placental malaria and maternal HIV.13 These factors may lead to reduced passive immunity in indigenous neonates and infants.
We aimed to explore whether differences in anti-pneumococcal, serotype-specific IgG contribute to the disparate rates of otitis media between Australian indigenous and Gambian infants. We evaluated the serum levels and functional capacity of anti-pneumococcal, serotype-specific IgG in cord blood in these 2 populations.
Cord blood samples were obtained from 36 Gambian indigenous and 34 Australian indigenous mothers who were not previously immunized with pneumococcal vaccine.
Gambian Cord Samples
Cord samples from The Gambia were collected as part of a previous study.14 Pregnant women attending the antenatal clinic at the Fajikunda Health Centre in Banjul were recruited and women with any pregnancy complications were excluded from the study. At birth, cord blood was collected, sera separated immediately and stored at −80°C at the Medical Research Council laboratory. Samples were transported by Air Express to Australia on dry ice and stored at the Murdoch Childrens Research Institute at −80°C until assayed.
Australian Indigenous Cord Samples
Cord samples from Australian indigenous mothers were collected as part of the PRIORiTi study.15 Mothers delivered at The Royal Darwin hospital and at birth, cord blood was collected, sera separated immediately and stored at −80°C at the Menzies School of Health Research in Darwin. Samples were transported to the Murdoch Childrens Research Institute in liquid nitrogen and stored there at −80°C until assayed. This study had ethics approval from the Gambian Government/MRC Laboratories Joint Ethics Committee, The Top End Human Research Ethics Committee, the Tiwi Health Board and the HREC at the Royal Children’s Hospital.
Measurement of Serotype-specific IgG and IgG Subclasses
Serotype-specific IgG, IgG1 and IgG2 were measured using a modified 3rd generation enzyme linked immunosorbent assay based on World Health Organization recommendations.16,17 Sample dilutions were analyzed in duplicate and 3 controls (low, medium and high) were included on each plate. Serotype-specific IgG was measured to serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 15B, 18C, 19F and 23F. Serotype-specific IgG1 and IgG2 were measured to serotypes 1, 6B, 19F and 23F.
These serotypes were chosen as serotype 6B, 19F and 23F were the common serotypes implicated in carriage, otitis media with effusion and invasive pneumococcal disease in both Australian indigenous populations18 and Gambian infant populations.19 Before the national introduction of a pneumococcal conjugate vaccine in The Gambia, a carriage study of 236 infants in 21 villages from birth to 1 year revealed that all infants carried S. pneumoniae at some time point and the 5 serotypes 6B, 19F, 6A, 14 and 23F accounted for 51% of the isolates (see Table, Supplemental Digital Content, https://links.lww.com/INF/B772). The serotype-specific IgG1 and IgG2 to serotype 1 was measured as this serotype is a substantial cause of invasive disease and was the cause of a community outbreak at the time the indigenous cord samples were collected, although is generally not identified as a prevalent colonizing serotype.20,21
The avidity of serotype-specific IgG, a surrogate of functional antibody activity,22,23 was determined using previously published methods.23–26 We have previously reported a direct correlation between antibody avidity and opsonophagocytic assays.27 Serotype-specific avidity was measured to serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 15B, 18C, 19F and 23F. Avidity indices were calculated as the percentage of bound antibody following sodium thiocyanate (0.5 M NaSCN) elution using an optical density interpolation value of 0.5 units (interpolation valueNaSCN/interpolation valuePBS/FCS) × 100.
Data were analyzed using GraphPad Prism version 5 (GraphPad Software Inc, CA). Serotype-specific antibody concentrations were log10 transformed and the geometric mean concentration (GMC) and 95% confidence intervals (CIs) determined. Serotype-specific IgG, IgG1 and IgG2 GMC comparisons were made using unpaired t-tests of the logged data. Group comparisons and the percentage of samples with a serotype-specific IgG > 1 μg/mL was calculated using Fishers exact test. It should be note that the result of this comparison was not altered when the cut-off for serotype-specific IgG was increased to 1.2 μg/mL.
Avidity was expressed as serotype-specific median avidity index (%) and interquartile range for each serotype. Group comparisons and the percentage of samples with an avidity index of > 50% was determined using Fishers exact test. As multiple comparisons were made, P < 0.02 was considered statistically significant.
Serotype-specific IgG and IgG Subclasses
The GMCs and 95% CIs for serotype-specific IgG in the cord sera samples are detailed in Figure 1. The serotype-specific IgG GMC in Australian indigenous cord sera were significantly greater than the GMC in Gambian indigenous sera for 6 of 12 serotypes (1, 4, 5, 7F, 14 and 18C; P < 0.02). A trend was seen for higher IgG GMC for serotype 19F in indigenous compared with Gambian cord samples (GMC = 1.39; 95% CI: 0.88 – 2.19) versus (GMC = 0.80; 95% CI: 0.62 – 1.02); P < 0.03.
Although mothers had not received prior pneumococcal immunization, a GMC >1 μg/mL was demonstrated in Australian indigenous cord sera for serotype 14 and 19F and for Gambian cord sera for serotype 14 (Fig. 2). In the absence of any history of maternal immunization, a cut-off of 1 μg/mL serotype-specific IgG was arbitrarily chosen as indicative of a protective level. A significantly greater percentage of Australian indigenous infants had protective titers (>1 μg/mL) of serotype-specific IgG for serotypes 4 and 5 compared with Gambian cord sera. Twenty-four percentage of Australian indigenous samples recorded titers > 1μg/mL for serotype 4 and 29% for serotype 5. In contrast, all Gambian infants had nonprotective levels of serotype-specific IgG (<1 μg/mL) for serotype 4 and all but 2 of 36 infants had nonprotective levels of serotype-specific IgG against serotype 5. More than 80% of Australian indigenous infants (29 of 34) and >60% of Gambian infants (23 of 36) had titers of >1 μg/mL against serotype 14.
Australian indigenous infants had significantly higher levels of serotype-specific IgG1 for serotypes 6B, 19F and 23F compared with the Gambian group (Fig. 3). Australian indigenous infants also had higher levels serotype-specific IgG2 against serotype 1 compared with the Gambian infants.
The median avidity index in indigenous cord samples was significantly reduced compared with Gambian cord samples for serotypes 1, 3, 4, 5 and 6B and significantly increased for serotype 7F and 9V. However, these results must be interpreted with caution as the serotype-specific IgG in a number of samples was low and the antibody avidity could not be measured (see Table, Supplemental Digital Content, https://links.lww.com/INF/B773).
The percentage of samples with an avidity index of > 50% is detailed in Figure 4 (results for serotype 1, 6B, 19F and 23F have been omitted as the antibody avidity could not be measured in > 30% of samples; see Table, Supplemental Digital Content, https://links.lww.com/INF/B773). The proportion of Gambian cord sera with an avidity index of > 50% was significantly lower than the proportion of Australian indigenous cord sera for serotypes 3, 4, 5 and 7F.
We have examined the passively acquired humoral immunity to pneumococcus in 2 selected populations with differing rates of chronic otitis media despite similar rates of pathogen colonization: Australian indigenous infants who demonstrate high rates of chronic otitis media and pneumococcal colonization in infancy and Gambian infants with high rates of colonization in infancy but low rates of chronic otitis media.
Indeed, in the Australian indigenous population presented here, by 2 months 72% of infants had experienced an episode of otitis media with effusion and by 6 months all infants enrolled in the study had experienced an episode of otitis media with effusion and 20% had tympanic membrane perforation.15 It is unclear why Gambian infants do not experience the high rates of otitis media when compared with Australian indigenous infants despite having early colonization of the nasopharynx.9 One possibility is a reduction in the serum levels and/or functional capacity of pathogen-specific antibodies, particularly passively acquired anti-pneumococcal antibodies from the mother, which have been considered to provide a major defense against bacterial infections in the early months of life. In this study, we found that cord blood levels of serotype-specific IgG and IgG subclasses were in fact higher in indigenous Australian infants when compared with Gambian infants for 6 of 12 serotypes, and a greater proportion of Australian indigenous infants had protective levels of serotype-specific IgG when compared with Gambian infants.
Moreover, Australian indigenous infants had significantly higher median avidity index for many serotypes tested (serotype 3, 4, 5, 7 and 9V) compared with Gambian infants, and a greater proportion of indigenous infants had an avidity index >50% for serotypes 1 and 6B. These findings indicate that the increased prevalence of otitis media among indigenous Australians does not relate primarily to deficient levels or function of serotype-specific antibodies.
Decreased levels of IgG in Gambian neonates could be explained by the high incidence (51%) of placental malaria in this population.28 However, despite the incidence of maternal hypergammaglobulinaemia, reduced placental transfer of IgG1, IgG2 and IgG4 and significantly decreased transfer of IgG to herpesvirus type 1, respiratory syncytial virus and Varicella-zoster virus, Okoko et al28 reported no significant difference in placental transfer of anti-pneumococcal IgG between mothers with and without placental malaria.
A previous study by Salazar et al29 demonstrated that low cord type 14 and 19F antibodies were independent predictors of early onset of otitis media and that low cord levels led to early onset otitis media which in turn predisposed to recurrent and chronic disease. The relationship between antibody and development of disease remained significant after controlling for other risk factors such as day care attendance, parental smoking, breastfeeding and sibling or family history. By contrast, we found that cord blood from indigenous Australians had significantly higher titers of serotype-specific IgG against 6 of the 12 serotypes tested, compared with cord blood from Gambian infants. Specific IgG to serotype 14 and 19F were higher in Australian indigenous cord sera compared with Gambian cord sera (P < 0.003 and P < 0.03), respectively. These conflicting findings may relate to the different populations studied. To our knowledge, there have been no previous studies of serotype-specific IgG subclasses and their relation to otitis media or invasive pneumococcal disease.
Our finding of increased avidity index of serotype-specific IgG among Australian indigenous infants is consistent with the findings of the Finnish otitis Media Vaccine Trial, which reported a lack of relationship between vaccine-specific antibody avidity postimmunization and protection against acute otitis media (AOM). However, increased antibody avidity maturation was associated with increased protection against AOM.30 This finding was confirmed when functional response was measured by opsonophagocytosis31 indicating that antibody concentration and functional antibody response postimmunization provided low clinical relevance for protection against AOM.
As has been reported previously,13 IgG1 subclass antibodies are preferentially transferred across the placenta compared with IgG2 and we have demonstrated significantly higher titers of IgG1 for 3 of 4 serotypes tested in indigenous cord samples compared with Gambian cord samples. Although Gambian mothers are frequently colonized with pneumococci, cord blood titers of serotype-specific IgG were generally similar to cord blood titers from Australian non-indigenous infants whose mothers are not commonly colonized (unpublished, data not shown). This suggests that colonization per se does not necessarily induce production of protective serotype-specific IgG; and conversely, that the high rates of infant colonization are not adequately explained by low titers of passively acquired maternal anti-pneumococcal antibody levels. The fact that colonization occurs in the first 3 months of life7 despite the presence of maternal serotype-specific antibody suggests there are other factors that may contribute to high colonization rates apart from the specific antibody response. In particular, the innate immune response may play a significant role. Nevertheless, there is little evidence to support a defect in innate immunity among either of the populations tested. A comprehensive study of toll-like receptor (TLR)-mediated innate immune responses in Gambian infants concluded that Gambian infants have “broadly similar toll-like receptor-mediated responses to those found in a Western European environment”.32 Jose et al33 examined a number of the elements of innate immune responses in young aboriginal children and found that immune responses were only depressed if the child was malnourished as compared with Caucasian children. Wiertsema and Leach34 reviewed the evidence concerning innate immunity and Australian aboriginal children and concluded further research was required as the contribution of the innate immune response to the prevention of otitis media was not well-understood.
Other factors may contribute to increased risk of otitis media in the Australian indigenous infants. For example, local immune responses in the nasopharynx may play an important role in immune protection, although Verhaegh et al35 examined both the local antibody response to M. catarrhalis and S. pneumoniae proteins in middle ear fluids in young Australian indigenous children from birth to 5 years of age with either recurrent AOM or chronic otitis media with effusion and found no association between either local or systemic antibody titers and clinical features. Another factor that might contribute to early colonization and infection is dysfunction of Eustachian tube drainage of the middle ear. Eustachian tube dysfunction as reflected in abnormal tympanograms is a common finding in aboriginal children. Jacoby et al36 found that 84% of aboriginal children had a type B tympanogram on at least 1 occasion. Leach et al7 postulated that early bacterial colonization may lead to Eustachian tube damage with consequent chronic otitis media. It is possible that Eustachian tube dysfunction might contribute to the observed high rates of early colonization and infection and further investigation of this is warranted.
The authors wish to sincerely thank all the families participating in these studies, the mothers from The Gambia and the Tiwi Islands, Australia for allowing their cord blood to be used and the staff in the laboratories at the Medical Research Council, Banjul, The Gambia Health and the Menzies School of Health Research, Darwin, Australia.
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