Acute otitis media (AOM) constitutes one of the most common ear infections caused by respiratory viral and bacterial pathogens in children. The common bacteria that cause AOM include Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pyogenes, which are detected in approximately 70% of the bacterial culture tests.1–5 Respiratory viruses were detected in <5% of the viral cultures before the 1970s, but their detection rate increased to 20% since the 1980s owing to new techniques for viral culture and antigen detection.4–7 In recent years, following the advent of molecular biology techniques such as polymerase chain reaction (PCR), respiratory viruses are detected in 32%–67%.6–12 However, only few reports have comprehensively examined the role of respiratory viruses11,12 and almost none have comprehensively and simultaneously examined and presented details on viruses and bacteria in the nasopharynx.
AOM is caused by interactions between an antecedent viral infection and colonized bacteria in the nasopharynx. Several studies have previously been published regarding how respiratory viral infection contributes to AOM as a complication of upper respiratory infection (URI) in children and the importance of viral–bacterial interaction in the nasopharynx.13–16 Chonmaitree et al13 performed a prospective, longitudinal study of 294 healthy children and showed that >60% of symptomatic upper respiratory URI episodes were complicated by AOM or otitis media with effusion. They also reported that AOM occurred in about half of children with URI associated with adenovirus (AdV), respiratory syncytial virus (RSV) or coronavirus and about one-third of those with influenza virus, parainfluenza virus (PIV), enterovirus or rhinovirus (RV). Xu et al14 reported that polymicrobial colonization afforded a high risk of AOM in the infection-prone children. Ruohola et al15 also showed that co-colonization of bacteria in the nasopharynx increased the risk of AOM in children and RSV might contribute to AOM even without bacterial colonization. Finally, Pettigrew et al16 studied the effect of respiratory viruses and bacterial coinfection, suggesting that individuals with both S. pneumoniae with RSV and H. influenza with human bocavirus (HBoV) were at high risk for AOM. These findings indicate the importance of examining for numerous types of respiratory virus and bacteria to understand the pathogenesis of pediatric AOM and how it becomes refractory.
In Japan, pneumococcal conjugate vaccine 7 (PCV7) was introduced in 2010 and PCV13 in 2013. Before this, RSV infection with colonization of S. pneumoniae indicated a high risk of pediatric AOM, whereas the microbiology of AOM has changed since PCV was introduced.16 We believe that the comprehensive PCR detection of respiratory viruses and bacteria in nasopharyngeal aspirates (NPA) and middle ear fluid (MEF) and their comparative examination will markedly contribute to our understanding of the microbiology of pediatric AOM and the optimal treatment of complicated AOM in the post-PCV era.
MATERIALS AND METHODS
Patients and Sample Collection
A total of 122 children who visited Sawada Eye and Ear Clinic, Kochi, Japan, from January 2016, to December 2017, were enrolled in this study of AOM. The median age of the study population was 14.5 months (range, 4–36 months). All children received PCV13. AOM was defined by an otolaryngologist (S.S.), after observing the otoscopic findings of MEF, bulging of tympanic membrane, and at least one of these symptoms: earache, tugging at the ear, fever, redness, rubbing and restless sleep. Children with recurrent AOM, chronic otitis media with effusion, tympanostomy tube or spontaneous perforation of tympanic membrane were excluded from the study. We enrolled patients with AOM presenting with the indication of paracentesis, prolonged fever or earache, or those referred from another clinic with treatment failure. Written informed consent was obtained from the parents of all children before enrollment. This study was approved by the Kanagawa Medical Practitioners Association (No. 15005).
The MEF and NPA samples were obtained during each child’s initial visit. The tympanic membrane was anesthetized with 4% lidocaine. The external ear canal was cleaned and paracentesis was performed with a lancet by an otolaryngologist. The MEF was aspirated into a sterile suction tip with 1 mL phosphate-buffered saline. The NPA samples were collected in a sterile suction tube, which was gently inserted into the child’s nose for sample collection by suction. Nucleic acid was extracted from the MEF and NPA samples and real-time PCR was immediately performed.
Bacterial Culture (MEF)
Bacterial culture was performed for all the MEF samples. Each sample was transported by BD culture swab (Beckton Dickinson, Sparks, MD) to BML Clinical Examination Laboratory (BML Inc., Kawagoe, Japan) for analysis. Each sample was plated onto sheep blood agar and chocolate agar, after which the plates were incubated at 35°C for 24 hours under 5% CO2. Pathogenic bacteria were identified using standard methods.
Virus and Bacteria Detection via Real-time PCR
Nucleic acid was extracted from 200 µL aliquots of the MEF and NPA samples using the QIAamp MinElute Virus spin kit (QIAGEN, Hilden, Germany). A CycleavePCR kit (TaKaRa Bio, Shiga, Japan; catalog number CY216) comprising reverse transcription (RT) reagent to produce cDNA from RNA was used for viral identification. Cycleave probes were labeled with a fluorescent reporter, 6-carboxyfluorescein (FAM) or carboxy-X-rhodamine (ROX), at the 50-end and with an eclipse dark quencher at the 30-end to search for 11 respiratory viruses (1 or 2 per well) as follows: RSV subgroup A (RSV-A) and RSV subgroup B (RSV-B); PIV type 1 (PIV1) and PIV type 2 (PIV2); PIV type 3 (PIV3) and human metapneumovirus (hMPV); influenza virus type A and type B; AdV and HBoV and RV. The PCR sensitivity was represented as approximately 10 plaque-forming units per well for respiratory viruses.17,18 The RT reaction was performed at 37°C for 15 minutes with the RT agent supplied in the Cycleave PCR kit.
Another CycleavePCR kit (catalog number CY214) was used to identify 6 bacterial pathogens: S. pneumoniae, H. influenzae, Mycoplasma pneumoniae, Chlamydia pneumoniae, S. pyogenes and Legionella pneumophila. The sensitivity of this kit has also been described to be about 10 colony-forming units per well.18,19 Before amplification, 2 µL of cDNA or DNA sample was added to the wells comprising the reaction mixture and maintained at 4°C until amplification began at 95°C for 30 seconds, followed by 40 cycles of PCR: 95°C for 15 seconds, 55°C for 30 seconds and 75°C for 15 seconds, using a Thermal Cycler TP900 (TaKaRa Bio).
Another bacterial PCR kit, FTD Bacterial pneumonia CAP (FastTrack Diagnostics, Sliema, Malta) was used for all the 17 MEF samples that did not yield bacteria by the Cycleave PCR kit to detect M. catarrhalis. This kit was used with the CFX96 thermal cycler (Bio-Rad, Hercules, CA).
Microbial Diversity of the MEF Samples
We examined all the MEF samples in the 122 cases of pediatric AOM by viral PCR, with bacterial culture or PCR. At least 1 respiratory viral or bacterial pathogen was detected from the MEF in 93 cases (76%) through bacterial culture and viral PCR (Fig. 1A) and in 120 cases (98%) through bacterial PCR and viral PCR (Fig. 1B). By bacterial culture and viral PCR, bacteria alone were detected in 30%, viruses and bacteria together in 31%, virus alone in 24% and no pathogen was detected in 15% of the MEF samples (Fig. 1A). From bacterial PCR and viral PCR, bacteria alone were detected in 43%, viruses and bacteria together in 48%, virus alone in 7% and no pathogen was detected in 2% of the MEF samples (Fig. 1B).
Viral Pathogens in MEF and NPA
Subsequently, we compared the detection of respiratory viruses between the MEF and NPA samples (Table 1). Respiratory viruses detected by PCR in MEF and NPA are listed in Table 1. Viral nucleic acid was present in MEF of 67 cases (55%) and in NPA of 84 cases (69%). RSV was the most common virus detected in MEF samples (21% with RSV-A and RSV-B combined; 1 case of hMPV was also detected), followed by PIV3 in 18 cases (15%), AdV in 10 (8%), RV in 6 (5%), hBoV in 4 (3%), influenza virus type A in 3 (2%) and hMPV in 1 (0.8% at the same time as RSV-B). All viruses detected in MEF (68 pathogens) were also present in NPA.
Bacterial Culture and Bacterial PCR Results of MEF Samples
We compared the bacterial culture and PCR results of MEF samples of all the pediatric AOM cases. At least 1 bacterial pathogen was detected from MEF by bacterial culture in 75 cases (62%) and by bacterial PCR in 112 cases (92%; Table 2). In bacterial culture, H. influenzae was the most common and was detected in 43 cases (35%), S. pneumoniae was detected in 19 cases (16%), and both H. influenzae and S. pneumoniae were detected in 3 cases (2%). In comparison, bacterial PCR detected bacteria in 112 cases (89%), with H. influenzae or S. pneumoniae detected in 104 cases (85%).
Viral and Bacterial PCR Results of MEF Samples
We examined the detection of respiratory viruses and bacteria in MEF by PCR (Table 3). Bacteria were detected in 89% of 122 cases, with H. influenzae detected in 55 (45%), S. pneumoniae detected in 24 (20%) and S. pneumonia + H. influenzae detected in 25 (20%). These 2 bacteria were detected in 104 cases (85.2%). In addition, M. catarrhalis was detected in 7 cases (6%) and M. pneumoniae in 1 case (0.8%). For each viral pathogen, no specific distribution of bacterial pathogens was observed in MEF.
The present study highlights 2 important findings: pediatric AOM constitutes a polymicrobial infection and multiplex PCR is suitable for the detection of multiple pathogens in patients with pediatric AOM. Results of the present study revealed more than 1 respiratory virus and/or bacteria in 98% of the cases. The detection sensitivity of the Cycleave PCR used for respiratory viruses and bacteria was high in the present study, which rendered it suitable for the detection of pathogenic microorganisms causing pediatric respiratory infections.17–19 The result of this study showed that the multiplex PCR kit was useful for the detection of respiratory viruses and bacteria in MEF and NPA in pediatric AOM.
Among detected viruses, RSV was the most common (21%), with the result supporting RSV as one of the most important respiratory viruses causing pediatric AOM. The detection of respiratory viruses before multidetection PCR through viral culture and antigen detection was ≤30%.3,4 However, Ruohola et al11 performed comprehensive PCR for respiratory viruses and bacteria in otorrhea samples from children with a tympanostomy tube and detected viruses in the MEF of 70% of the cases and both bacteria and virus in 66% of the cases. In the present study, viruses were detected in the MEF of 55% of the cases, and both bacteria and viruses were detected in 43% of the cases, which were similar to the data obtained by Ruohola et al.11 However, the ratio of viruses detected in our study differed from that detected in their report, possibly owing to the differences in patient age (the patients in our study were marginally younger), cases (the patients in the study by Ruohola et al11 had a tube inserted) and the types of viruses detected. In addition, their study period was ≤1 year, whereas we accumulated cases from ≥2 years to detect epidemics.
Bacteria were detected in 62% of the cases by bacterial culture and 89% of the cases by bacterial PCR in the present study, with H. influenzae and S. pneumoniae being the most commonly identified. Using bacterial culture, H. influenzae was the most frequently detected at 38% and S. pneumoniae was detected at 18% of MEF specimens. In the pre-PCV era, the average rate of H. influenzae was 22.2% and that of S. pneumoniae was 26.4% by bacterial culture in Asian patients with pediatric AOM.16 In the present study, all patients had received PCV. In the post-PCV era, we confirmed H. influenzae comprised the most frequent bacterial pathogen in pediatric AOM. In a recent review, PCR analysis increased the frequency of detection of H. influenzae and S. pneumoniae by 3.2-fold compared with that from culture.20 Our data support multiplex PCR as a useful tool for the detection of respiratory bacteria from MEF samples of patients with pediatric AOM. In the present study, viral AOM without any bacteria was present only in 11% of the cases. Therefore, coinfection of viruses and bacteria is common when detecting respiratory microorganisms comprehensively using molecular techniques. In some cases, multiple types of viruses were detected, further confirming that pediatric AOM is a polymicrobial infection.
In pediatric AOM, viral and bacterial coinfection is considered to enhance inflammation and can lead to prolonged symptoms and adverse clinical outcomes.4,7,21 The reasons for clinical failure may be the production of inflammatory mediators by viral infections,6,16,22–25 delayed clearance of bacteria caused by Eustachian tube dysfunction,26,27 polymorphonuclear leukocyte dysfunction due to viral infections28,29 and reduced penetration by antimicrobial agents.30 Therefore, the efficacy of an antibacterial agent is considered to be lower for AOM caused by coinfection with bacteria and viruses than for AOM caused by bacteria alone. Hence, the simultaneous detection of viruses and bacteria from the middle ear is important for the treatment of AOM. In particular, pediatric AOM is associated with several types of viruses, and the role of the interactions between each virus and bacteria in causing infections requires further analysis through study of a large number of cases.
Another important finding of the present study is that we performed simultaneous and comprehensive detection of respiratory viruses in both the MEF and NPA. In AOM, as viral infection in the nasopharynx is involved in the pathogenesis of AOM, it is important that the detection from NPA and MEF specimens is carried out simultaneously. A few studies9,12 have detected various respiratory viruses simultaneously from both the MEF and NPA. Pitkäranta et al9 attempted to detect RV, RS virus and coronavirus in the MEF and NPA and detected the viruses in the MEF of 48% of the cases. However, the detected viruses were only of 3 types and the viruses in the MEF were not detected in the NPA in some reported cases.9 Yatsyshina et al12 reported comprehensive and simultaneous PCR study of respiratory viruses and bacteria from MEF and NPA. In their study,12 the detection rate of respiratory viruses from MEF was low (14.5%) and the concordance rate with a virus in the nasopharynx was also low; thus, they were unable to examine the association between infection of the nasopharynx by respiratory viruses and AOM sufficiently. In the present study, a respiratory virus was detected in NPA and MEF in 69% and 55% of the cases, respectively. In MEF, 1 type of virus was detected in 57 cases, 2 types in 9 cases and 3 types in 1 case. All these viruses were detected in the NPA samples. The result confirmed that all the viruses detected in the middle ear originate in the nasopharynx, where the respiratory viruses migrate to the middle ear through the Eustachian tube, causing AOM.
Our data suggest that multiple detection by real-time PCR is useful for the comprehensive detection of respiratory viruses and bacteria in pediatric AOM. Viruses detected in the MEF were all confirmed in the NPA, enabling establishment of the microbiologic etiology. For further understanding, we aim to acquire evidence from additional cases, investigate clinical outcomes for each pathogen and compare single and polymicrobial infections in pediatric AOM.
The present study had some limitations. The real-time PCR kit used for detecting respiratory viruses in the study does not detect coronavirus and enterovirus; therefore, these microorganisms were not detected. In terms of differences with other studies,9,11 it was possible to detect certain types of viruses and bacteria as discussed above; however, the types of viruses and bacteria that can be detected will vary according to age. The present study examined causative viruses of AOM in children ≤3 years of age; thus, the results for pathogens in children ≥4 years of age are likely to be different.
The authors thank Sachie Ichikawa and Kana Osaki for their work in sample processing and Dr. Yoshuke Kamide for his appropriate advice.
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