Otitis media (OM) is one of the most frequently diagnosed bacterial infections in children worldwide.1 With peak incidence occurring within the first 24 months of life,2 OM is a leading reason for antibiotic prescriptions in children in the United States.3 The clinical spectrum of OM ranges from asymptomatic to early uncomplicated to complex disease such as recurrent, nonresponsive, dry perforation, spontaneously draining and chronic suppurative OM.4 The 2 leading causative agents of OM are Streptococcus pneumoniae (Spn) and nontypeable Haemophilus influenzae (NTHi), which have also been implicated as major causes of other severe infections.5,6Staphylococcus aureus, Pseudomonas aeruginosa and Moraxella catarrhalis have also been implicated but to a lesser extent.4,7
However, most of the currently available data on the specific roles of these organisms in the burden of OM have been generated from children in high-income countries, and there is paucity of data from developing nations like Bangladesh and other South Asian countries. Although 2 studies have shown that OM is a significant cause of morbidity in Bangladesh,8,9 no systematic study has been performed to assess the pathogens responsible for OM here.
In 2006, noting the enormous global burden of Spn-associated morbidity and mortality, the World Health Organization recommended the inclusion of pneumococcal conjugate vaccine (PCV) in routine immunization programs of all countries and assessment of its impact on pneumonia, other invasive diseases and OM.10 In March 2015, Bangladesh introduced the 10-valent PCV (PCV10), which was designed to provide protection against 10 serotypes: 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F; in addition, cross-protection against 6A and 19A is anticipated because of their immunologic similarities with vaccine serotypes 6B and 19F and the conjugation method.11,12 PCV10 is conjugated to a recombinant form of protein D of NTHi, with the expectation that the vaccine will also provide protection against NTHi; however, most studies do not provide support that cross protection against NTHi occurs in practice.6,13–17
To generate data on OM in Bangladesh, our group initiated a surveillance study to identify and characterize the bacterial pathogens of OM in the ear, nose and throat (ENT) clinic of the outpatient department of Dhaka Shishu Hospital (DSH) in April 2014. The objectives were to determine (1) the number of OM cases at the largest pediatric hospital of the country, (2) the pathogens in OM cases with otorrhea (discharge), (3) the antimicrobial susceptibility of Spn and Hi isolates from OM cases and (4) the serotype distribution of Spn and Hi.
This was a prospective surveillance study conducted from April 2014 to March 2015.
Study Site and Population
The study was run at DSH, the largest pediatric hospital in Bangladesh, which serves children from 0 to 18 years old. This hospital has a separate OPD for treatment and management of all cases seeking care for OM and other ENT problems. On average, 612 children visit the ENT OPD of DSH per month.
Case Identification and Enrollment Criteria
Children with suspected OM were identified by a study physician based on World Health Organization–recommended clinical criteria and otoscopic findings.18,19 In brief, the inclusion criteria for OM cases were presence of (1) acute onset of 1 or more sign/symptoms of infection such as fever, earache, ear tugging or irritability and (2) middle ear inflammation with/without middle ear effusion or spontaneous discharge (otorrhea). From enrolled children with/without otorrhea (not caused by otitis externa), sex, age, duration of discharge, if present, and history of antibiotic usage in the previous 7 days were noted. Specimens were collected from the enrolled cases with otorrhea after obtaining informed consent from the caregiver.
If the children with OM presented with otorrhea, an ear swab was collected after removal of debris from the external auditory canal by a trained research assistant. Specimens were collected using a flocked swab and were placed into a tube containing STGG medium (2% skim milk, 3% tryptone, 10% glycerol and 0.5% glucose). Each specimen was then cultured within 3 hours in the laboratory and examined for the presence of bacterial pathogens.
All specimens were cultured on blood agar with and without gentamicin, chocolate agar with bacitracin and MacConkey agar.18 We examined these selective media for the presence of Hi and Spn (in 5% CO2 environment) and also identified other bacterial pathogens, including S. aureus, M. catarrhalis, Pseudomonas spp., etc. Pseudomonas spp. were further classified using the analytical profile index for non-enterobacteriaceae.
Serotyping of pneumococcal isolates was performed using quellung reaction; serotyping of Hi was conducted using a previously established polymerase chain reaction method.20,21 Antibiotic susceptibility of all Spn and Hi strains was screened using the disc diffusion method, and the results were interpreted based on the most recent Clinical and Laboratory Standards Institute (CLSI) guidelines.22 Penicillin (1 μg oxacillin disc), trimethoprim-sulfamethoxazole (1/19, 25 μg disc), chloramphenicol (30 μg disc), erythromycin (15 μg disc) and ciprofloxacin (5 μg disc) were used for Spn. Nonsusceptible Spn isolates, displaying intermediate or complete resistance to penicillin, trimethoprim-sulfamethoxazole and erythromycin, were subjected to E-test for de termination of minimum inhibitory concentrations. Antibiotic susceptibility testing of Hi isolates was performed for ampicillin (10 μg disc), trimethoprim/sulfamethoxazole (1/19, 25 μg disc), chloramphenicol (30 μg disc), erythromycin (15 μg disc), ciprofloxacin (5 μg disc), ceftriaxone (30 μg disc) and tetracycline (30 μg disc).
Data were analyzed to elucidate the age distribution of OM cases, the pathogens associated with them, serotype distribution and antibiotic susceptibility profiles of Spn and Hi isolates. Common contaminants were excluded.
Vaccine serotype coverage was calculated based on the serotypes targeted by the PCV10 formulation and by PCV10 + 6A and PCV10 + 6A+19A. Mann–Whitney test was used to analyze the significance of difference between median duration of otorrhea and pathogen detected.
This surveillance protocol was approved by the Institutional Review Board of Bangladesh Institute of Child Health, DSH. The US Centers for Disease Control determined this project to be public health practice not requiring Centers for Disease Control Institutional Review Board review. Specimens were collected as part of routine patient care.
From April 2014 to March 2015, 7342 children visited the ENT-OPD of DSH, all of whom were screened for OM by the study physician (Fig. 1). In total, 1111 OM episodes were recorded based on the defined criteria, and all cases were enrolled in this study; 45% (496/1111) of the episodes were in girls. Otorrhea was seen in 88% (981/1111), and the duration of otorrhea ranged from 1 day to 18 months; median duration was 5 days. Specimens were successfully collected from 91% (891/981) of these cases; specimens were not collected from children who suffered from excessive ear pain or whose caregivers did not provide consent (9%, 90/981). Enrolled children were between 0 and 14 years of age (Fig. 2A), and the median age was 23 months (2 years). One-third (32%; 351/1111) of the episodes were observed in the first year of life. Within the first year, the highest proportion of cases was seen in children between 4 and 10 months of age (24%; 271/1111; Fig. 2B).
Specimens were successfully collected from 891 (91%) of 981 children with otorrhea. Fifty-one percent (452/891) of specimens were culture positive for at least 1 pathogen (Supplementary Table 1, http://links.lww.com/INF/D154). Overall, Hi was the most frequently isolated organism (21%, 187/891), followed by Spn (18%, 164/891), S. aureus (9%, 83/891) and P. aeruginosa (4%, 38/891). While 79% (356/452) of culture-positive specimens contained a single pathogen, 2 pathogens were detected in 20% (89/452) and 3 in 2% (7/452) of the cases. In cases with mixed pathogens Hi was the predominant organism, being present in 78% (75/96) of the cases. Spn was present in 72% (69/96) of mixed-pathogen cases, and Spn and Hi occurred together in 60% (58/96) of cases.
Although there were differences in prevalence of the pathogens in otorrhea cases, no significant difference between the presence of each of the 4 predominant pathogens and median duration of otorrhea was found (Fig. 3).
Serotype Distribution of S. pneumoniae and H. influenzae and Expected Vaccine Coverage
Ninety-eight percent (56/57) of Hi isolates serotyped were nontypeable (NTHi), and the remaining 1 isolate was Hi type f. On the other hand, 36 different pneumococcal serotypes were detected in this study (Fig. 4). The most common serotype was 19A (16%, 26/164), followed by 19F (14%, 23/164), 3 (8%, 13/164) and 14 (7%, 12/164). Taking the serotype distribution into account, the expected coverage of PCV10 for pneumococcal OM was 46%; expected coverage of PCV10 + 6A was 49%. If protection against 19A was achieved, the coverage of PCV10 + 6A+19A would be 65%. For all OM cases, expected coverage of PCV10 was 8%, PCV10 + 6A 9% and PCV10 + 6A+19A 12%.
Antimicrobial Susceptibility Testing of S. pneumoniae and Nontypeable H. influenzae
Overall, 16% (27/164) of the Spn isolates were susceptible to all antibiotics, while 30% were nonsusceptible to 1, 49% to 2 and 4% to 3 different antibiotics tested (Table 1). Ninety-nine percent (163/164) of the strains were susceptible to penicillin, while 77% (126/164) were nonsusceptible to trimethoprim-sulfamethoxazole and 58% (95/164) nonsusceptible to erythromycin.
Ninety-one percent (69/76) of the PCV10 isolates were nonsusceptible to at least 1 antibiotic, compared with 77% (68/88) of isolates of nonvaccine serotypes (P = 0.02; Table 1). Serotype 19A was found to be the most nonsusceptible serotype; 92% (24/26) of 19A isolates were nonsusceptible to trimethoprim-sulfamethoxazole, and 100% (26/26) were nonsusceptible to erythromycin; all 19A isolates were susceptible to penicillin.
Of all the NTHi isolates, 3% (5/187) were susceptible to all antibiotics and 18% were nonsusceptible to 1, 32% to 2 and 18% to 3 different antibiotics. Although no NTHi isolates were nonsusceptible to ciprofloxacin or ceftriaxone, 92% (172/187) were nonsusceptible to erythromycin, 67% (126/187) to tetracycline, 41% (76/187) to trimethoprim-sulfamethoxazole, 23% (43/187) to ampicillin and 16% (31/187) to chloramphenicol.
History of Antibiotic Consumption by Cases
Of the 891 cases from whom specimens were collected, we were able to obtain information on antibiotic consumption in the preceding 7 days from 890 cases. In total, caregivers reported antibiotic use for 2% (17/890) of children with OM. Antibiotic exposure was reported in 0.7% (3/451) of children with culture-positive specimens, as compared with 3% (14/439) of children with culture-negative specimens (P = 0.006).
Here, we report the first dataset on pathogens of OM in Bangladesh and, to our knowledge, the largest series in South Asia. The data will help institute evidence-based treatment and prevention policies in Bangladesh and in the surrounding region.
From April 2014 to March 2015, 15% of the 7342 children seeking care at the ENT-OPD of DSH presented with clinical manifestations of OM. Children of 2 years old or younger accounted for half of all disease episodes, a finding that corroborates with those of other studies.1,2 Overall, 88% of children with OM presented with otorrhea, a proportion that is higher than has been observed in most other studies.23 This high rate of otorrhea in OM children and very few nonotorrhea cases recorded in our surveillance study may be explained by poor care–seeking behavior in Bangladesh24; with no national healthcare system in place, patients pay out of their pockets and sick people often do not come to receive treatment in facilities unless the symptoms are severe like ear discharge. This also suggests that the number of cases seen in the hospital is an underestimation of the total burden of OM among children.
Of the 891 specimens collected, only 51% were culture positive. This is lower than seen in other studies.25,26 As specimens were only collected from cases with otorrhea and a large number of these cases had discharge for several days, our surveillance may be biased toward complex, chronic cases, where growth of biofilms is common.4 Hence, it is plausible that our culture methods were not able to detect pathogens in biofilms. Furthermore, OM can also be caused by viruses, and we did not attempt to detect viral pathogens in this study; it is plausible that some culture-negative OM cases were caused by viruses like Respiratory Syncytial Virus.27
The most commonly isolated organisms were NTHi (21%), Spn (18%), S. aureus (9%) and P. aeruginosa (4%)—pathogens that have been commonly associated with OM.28M. catarrhalis, which has been frequently isolated among samples in other studies, was only found in 0.4% of cases here.4,29 Eleven percent of the cases were polymicrobial, with 2 or 3 pathogens identified. Previous studies have suggested that Spn is more frequently responsible for early episodes of OM and may lead to subsequent polymicrobial infections, which are generally associated with NTHi.29
While 98% of H. influenzae were nontypeable, we identified 36 different serotypes of Spn (N = 164). Serotype 19A was the most common serotype, followed by 19F, 3, 14, 6B and 23F; 19A, 3 and 6A are not included in the PCV10 formulation and together were responsible for 32% of pneumococcal cases. The serotype distribution of pneumococci isolated from OM cases was different from that reported for invasive pneumococcal diseases (IPD) in Bangladesh. A multiyear surveillance study on IPD in Bangladesh identified serotypes 2, 1, 6B, 14 and 5 as the predominant causes, but these serotypes were seen less frequently (33/164) among the OM cases here.5 It is important to note that 19A is rarely seen among invasive cases (4%, ranking 9th),5 although it is the predominant serotype in OM cases.
Based on our findings, the serotype coverage of PCV10 was 46% and PCV10 + 6A was 49% for pneumococcal OM and 8% and 9% for all OM cases in this series. However, several clinical trials and pneumococcal vaccine impact studies conducted in Israel on OM have shown greater impact in practice than in theory.4,30 Previous vaccine impact studies conducted in Bangladesh have also revealed higher than theoretical vaccine impact for other syndrome.31,32 Thus, the coverage rate reported here may be an underestimate. On the other hand, however, there are reports of replacement disease by nonvaccine serotypes after PCV introduction; high rates of replacement OM by non-PCV10 serotypes can jeopardize the impact of the vaccine, which is difficult to predict beforehand. Hence, it is imperative to continue the ongoing surveillance and carefully monitor trends in serotype distribution.
Spn serotype 19A became an increasingly common cause of IPD in some countries after the introduction of PCV7, which lacks the 19A antigen.33–37 Some studies have suggested that the 19F antigen, included in PCV10, can provide cross-protection against 19A disease because of their immunologic similarities, but the magnitude of this protection is yet to be discerned and might depend on several factors in a given setting.12 Hence, it is difficult to accurately predict the impact of PCV10 introduction on 19A in Bangladesh. Furthermore, the H. influenzae protein D is used as a carrier protein in the PCV10 formulation to provide cross-protection against NTHi. However, the protective efficacy of PCV10 against diseases caused by NTHi is yet to be fully comprehended.6 Considering the high burden of NTHi OM in Bangladesh, it is important to continue monitoring impact in the post-vaccine era.
Several studies from the United States and countries in Asia and Europe have reported high rates of antibiotic nonsusceptibility, specifically to penicillin, among pneumococcal isolates. This has significant implications for treatment and in replacement/unmasking diseases after PCV introduction.38–41 However, this trend of resistance is not common in Bangladesh and India for either IPD or OM isolates.5,42 Here, we found that most Spn isolates were resistant to trimethoprim-sulfamethoxazole (77%) and erythromycin (58%); while resistance to trimethoprim-sulfamethoxazole was previously reported in 2003, a rise in erythromycin resistance is a more recent phenomenon.20,43 If trimethoprim-sulfamethoxazole and erythromycin were to be excluded from this analysis, 91% (150/164) of the strains would be susceptible. Remarkably, 99% of the strains were susceptible to penicillin and can be used for successful treatment. We also found that while most strains of NTHi were nonsusceptible to erythromycin and tetracycline, all strains were susceptible to ciprofloxacin and ceftriaxone.
The results of this study should be considered within the context of some limitations. First, tympanocentesis was not performed on OM children without otorrhea, and hence, the pathogens involved in such cases could not be detected. However, as 88% of the suspected OM cases presented with otorrhea, this limitation had a relatively small if any effect on our data. Furthermore, the possibility of missing bacterial pathogens in this study was low as we used selective and enriched (nonselective) media specifically for Spn and NTHi. However, as we did not assess presence of viruses in the specimens as discussed above, our study missed viruses, both as pathogens and copathogens. Another limitation of this study is that the surveillance was performed for only a length of 1 year, and hence year-to-year variations remain unknown. Finally, the surveillance reporting is not population based; it does not have a denominator and thus does not allow for incidence calculation. However, we are currently working to overcome this by combining the existing laboratory data with data from community-based surveillance of utilization of our study hospital to generate incidence estimates. Furthermore, previous work on impact of PCV has shown that for studies with large numbers of cases such as ours, it is possible to monitor vaccine impact in hospital-based surveillance systems in the absence of population-based studies.44
This is the first report on elucidation of pathogens of OM in Bangladesh and will contribute to the design of evidence-based treatment and prevention guidelines. We demonstrate that Spn is indeed a dominant pathogen found in OM cases in Bangladesh, and our data suggest that introduction of PCV10 will have substantial impact on the burden of OM in Bangladesh. We have established ongoing surveillance and are monitoring impact of PCV10 on OM. Based on the number of OM cases identified in our hospital and the high prevalence of Spn serotype 19A and NTHi, this ongoing surveillance will help us monitor the impact of PCV10 on overall pneumococcal and nonpneumococcal OM and also to estimate PCV10’s ability to protect against OM caused by vaccine-related serotypes 6A, 19A, as well as NTHi.
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
We are grateful to Rajib Chandra Das and Afiour Rahman for assistance with statistical analysis and to Dr. Asif Sattar, Zillur Rahman, Palash Mozumder and Shahina Tarafder for technical help.
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