Contents lists available atScienceDirect
International Journal of Pediatric Otorhinolaryngology
journal homepage:www.elsevier.com/locate/ijporlIncreased anti- EBV VCA IgG antibody levels are associated with need for
surgery in patients developing upper respiratory tract complications
Hande Arslan
a,∗, Tuba Çandar
b, Özge Vural
caDepartment of Otorhinolaryngology, University of Health Sciences, Dışkapı Yıldırım Beyazıt Training and Research Hospital, Ankara, Turkey bDepartment of Biochemistry, Ufuk University Faculty of Medicine, Ankara, Turkey
cDepartment of Pediatric Hematology, Trabzon Kanuni Training and Research Hospital, Trabzon, Turkey
A R T I C L E I N F O
Keywords: EBV
Adenoid hypertrophy Otitis media with effusion
A B S T R A C T
Introduction: The immune reaction developing against Ebstein-Barr virus (EBV) infection may be one of the major determinants of severe adenoid hypertrophy (AH) and chronic otitis media with effusion (COME) needing surgery. In this study, we aimed to investigate the relationship between these antibodies and the need for surgery due to complications such as severe AH and COME.
Methods: Sixty consecutive patients < 15 years old who were admitted to our outpatient clinics between January 2014 and December 2015 with severe AH ± COME and underwent adenoidectomy ± ventilation tube insertion and 129 control patients who had a history of EBV infection at least three months before the inclusion to the study without current symptoms of upper airway obstruction and middle ear disease were included in this study. Two groups of patients and a control group were studied: a) children who underwent adenoidectomy alone with no middle ear disease (group 1), b) children with COME and AH who underwent adenoidectomy and tympanostomy with ventilation tube insertion (group 2), and c) control group without adenoid hypertrophy or otitis media with effusion.
Results: Patients who needed surgery (Group 1 and 2) had significantly higher levels of anti-EBV VCA IgG antibodies than control patients (19.8 ± 16.4 vs. 1.7 ± 0.8 S/CO, p < 0.001). Anti-EBV VCA IgM levels did not differ between groups. Group 2 patients had also higher levels of Anti-EBV VCA IgG antibodies than group 1 patients (35.8 ± 16.7 vs. 11.8 ± 8.5 S/CO, p < 0.001). ROC curve analysis resulted in a cut-off point of 2.92 S/CO level for anti-EBV VCA IgG antibodies for need for surgery in EBV infected patients with 97% sen-sitivity and 98% specificity.
Conclusion: Markedly increased serum anti-EBV VCA IgG antibodies in children who developed upper re-spiratory tract complications such as severe AH and COME may show the significant role of enhanced immune system reaction in the pathogenesis of these complications due to EBV infection.
1. Introduction
The presence offluid in the middle ear without signs or symptoms of acute ear infection is called as otitis media with effusion (OME) [1]. 50% of children in thefirst year of their lifes experience OME, and by 2 years old 60% of children have one episode of OME [2]. When OME persists for 3 months from the date of onset (if known) or from the date of diagnosis (if onset is unknown) chronic OME (COME) occurs. 25% of OME episodes persist for 3 months and may cause hearing loss, beha-vioral problems or less often structural changes in tympanic membrane which may only resolve with surgery [3].
Adenoid hypertrophy (AH) is a common risk factor for COME in children. AH effects children with distinct nasal blockage or chronic
adenoditis [4]. It is recommended to use tympanostomy tubes when surgery is performed for OME in a child < 4 years old; adenoidectomy should not be performed unless a distinct indication exists (nasal ob-struction, chronic adenoiditis); but in children≥4 years old; it is re-commended to use tympanostomy tubes, adenoidectomy, or both when surgery is performed for OME and the clinician should document re-solution of OME, improved hearing, or improved quality of life when managing a child with OME [1].
Traditionally, OME leads to loss of ventilation and pressure equal-ization in the middle ear due to adenoidal hypertrophy and blockage of the Eustachian tube, but nowadays the importance of these factors di-minished to be considered as minor factors [5]. The biofilm activity in the adenoid and middle ear which produces a cascade of immune
https://doi.org/10.1016/j.ijporl.2018.05.032
Received 3 March 2018; Received in revised form 26 May 2018; Accepted 28 May 2018 ∗Corresponding author.
E-mail addresses:[email protected](H. Arslan),[email protected](T. Çandar),[email protected](Ö. Vural).
Available online 30 May 2018
0165-5876/ © 2018 Elsevier B.V. All rights reserved.
reaction causing inflammation after acute upper respiratory tract in-fection is now considered to be the most important factor in the ethiology of OME.
The Ebstein Barr virus (EBV) is a gamma herpes virus, whose genome consists of a linear double-stranded DNA. EBV is a widespread virus and it is estimated 90% of adults have come into contact with EBV and have positive antibodies. EBV is also a well-known virus that has role in both acute and chronic inflammation and it is associated with lymphoproliferative diseases.
Infection with EBV results in the development of virus specific an-tibodies. These antibodies are formed to structural protein or viral capsid antigens (VCAs), nonstructural proteins expressed early in the lytic cycle or early antigens (EAs), and nuclear proteins expressed during latent infections or EBNAs. VCAs arise early in the course of the illness and are seen in presentation in most cases. Anti-EBV VCA Ig G is the major diagnostic utility. After recovery, detectable titers of anti-EBV VCA Ig G antibody are maintained for life. Life-long persistence also makes Ig G VCA an epidemiological tool. Ig M antibodies are sensitive and specific for acute EBV infection. Titers of Ig M antibodies fall ra-pidly in 4–8 weeks after diagnosis [6].
To the best of our knowledge, a simultaneous analysis of anti-EBV antibodies in OME patients in their serum and ear effusion samples has not been previously reported in the literature. Besides, increased EBV IgG antibodies may suggest an exaggerated immune response to EBV infection in the ethiogenesis of COME. In this study, our aim was to determine the levels of these antibodies and suggest a relationship be-tween anti-EBV antibodies and diseases due to EBV infection, i.e. AH and COME needing surgical intervention.
2. Materials and methods
The present study was approved by the Institutional Review Board of Ufuk University Medical School with decision number 311020137 in October 2013 and parents of all patients signed informed consent be-fore entering the study.
2.1. Patient selection
All of the included individuals were selected from the patients who had known EBV infection documented by anti-EBV VCA IgG positivity. Sixty consecutive patients < 15 years old who were admitted to our outpatient clinics between January 2014 and December 2015 with the diagnosis of AH ± OME needing adenoidectomy ± ventilation tube insertion and 129 children who had a history of EBV infection at least > 3 months before the inclusion to the study without current symptoms of upper airway obstruction and middle ear disease were included in this study. Patient and control groups were known to have anti-EBV VCA IgG antibody positivity and the control group constituted children who had negative anti EBV VCA IgM values.
Children with craniofacial anomalies, hereditary or other systemic diseases, asthma, allergic diseases, nasal septal deviation, sinonasal infection, acute middle ear infection, tonsillar hypertrophy, chronic adenotonsillar infection and history of previous tonsillectomy ± adenoidectomy ± VT insertion were excluded from the study.
AH was diagnosed by an otolaryngologist in routine clinical practice based upon symptoms, signs and nasal endoscopy. Severe AH was de-fined at nasal endoscopy by the presence of adenoid in contact with the torus tubaris (grade 3) and/or vomer (grade 4) [7].
After physical examination of the ear and obstructive wax was cleaned, and tympanometry (AZ 26 Clinical Audiometer; Interacustics, Assens, Denmark) was performed. The instrument with a probe fre-quency of 226 Hz and air pressure range of−400 to +100 mm H2O was used for obtaining tympanograms. Tympanometric curve results were classified as types A, As, B or C [8]. Tympanogram with sharp peak and normal middle ear pressure was called type A, tympanogram
with reduced peak height and normal middle ear pressure was called type As, tympanogram with a low static admittance andflattened was called type B and tympanogram with highly negative peak pressure was called type C.
The decision of indication for adenoidectomy ± ventilation tubes was made according to the“Clinical Practice Guideline: Otitis Media with Effusion (Update)” guidelines [1]. We performed tympanostomy tube insertion with adenoidectomy to children with bilateral OME for ≥3 months (COME) and nasal obstruction. We performed adenoi-dectomy alone to children with nasal obstruction or recurrent sinonasal infections without COME.
Two groups of patients and a control group were studied: a) chil-dren who underwent adenoidectomy alone with no middle ear disease (group 1), b) children with COME and AH who underwent adenoi-dectomy and tympanostomy with ventilation tube insertion (group 2), and c) control group without adenoid hypertrophy or otitis media with effusion.
2.2. Laboratory measurements and clinical samples
Blood samples for biochemical parameters were taken from all participants after at least 8-h overnight fasting. Complete blood count parameters were measured at the simultaneous blood samples and were analyzed with hematology analyzer (CELL-DYN Ruby Hematology System, Illinois, USA).
Serum anti-EBV VCA IgM and IgG were measured at the simulta-neous blood samples and ear effusion samples were analyzed with Architect EBV VCA IgM and IgG Reagent Kit (Abbott, Germany).
Middle ear effusion fluid was obtained from patients under general anesthesia after the external auditory canal was washed and tympa-nostomy done. The collected effusion fluids and blood samples were immediately transferred to an Eppendorf tube and stored at−80 °C.
The Architect system calculates each result by using the ratio of the sample RLU to the cutoff RLU (S/CO). The criteria for interpretation of individual parameters were as follows: for VCA IgM, < 0.50 RLU was considered negative, 0.50 to 1 RLU grayzone, and > 1 RLU positive; for VCA IgG, < 0.75 RLU was considered negative, 0.75 to 1 RLU grayzone, and > 1 RLU positive.
2.3. Statistical analysis
The analysis of the results was performed using the IBM SPSS Statistics (Armonk, New York, USA) version 21.0 software for Windows. Data were tested for normal distribution using the Kolmogorov-Smirnov test. The categorical variables were shown as numbers of cases with percentages and the normally distributed continuous variables were shown as mean ± standard deviation. Median values (minimum-maximum) were used for the continuous variables which were not normally distributed. The Student's t-test was used for the analysis of the continuous variables which were normally distributed and theχ2 test for the categorical variables. Mann-Whitney U test was used for the variables which were not normally distributed. When > 2 groups were compared, differences among the groups were tested using ANOVA for normally distributed variables or the Kruskal-Wallis H test for non-normally distributed variables, and Fisher's exact test or Pearson's chi-square test for categorical variables. An optimal serum anti-EBV IgG level cut-off value for the detection of AH and COME was determined by receiver-operating characteristics analysis after comparing sensi-tivity and specificity at different cut-off values. A p value of 0.05 was considered statistically significant. Subsequent pair-wise comparisons were performed by Tukey's HSD post hoc analysis. For post-hoc tests p < 0.017 was considered significant. Pearson and Spearman correla-tion coefficients were used for correlacorrela-tion analysis where appropriate.
3. Results
Sixty [32 males (53.3%), mean age: 8.3 ± 5.7] consecutive patients who underwent adenoidectomy ± VT and 129 age and sex-matched healthy controls [68 males (52.7%), mean age: 7.7 ± 3.7] were en-rolled in our study. There was no significant difference between two groups in terms of gender (p: 0.9) and age (p: 0.9).
In group 1 (adenoidectomy), 38 (95%) patients had type A tympa-nogram and 2 (5%) patients had type C tympatympa-nogram. In group 2 (adenoidectomy + ventilation tube insertion) there were 16 (80%) patients with type B tympanogram and 4 (20%) patients had type C tympanogram. In control group (group 3), all participants had type A tympanograms (p < 0.01).
In group 1, 20 (50%) patients had grade 3 and 20 (50%) patients had grade 4 adenoid hypertrophy. In group 2, 14 (70%) patients had grade 4 and 4 (20%) patients had grade 3 and 2 (10%) patients had grade 2 adenoid hypertrophy. In control group, 63 (49%) participants had grade 1 and 66 (51%) participants had grade 2 adenoid hyper-trophy (p < 0.01).
Comparison of blood plasma values are summarized in Table 1. There were no statistically significant difference between groups in white blood cell, hemoglobin, C-reactive protein, aspartate transami-nase, alanine transamitransami-nase, blood urea nitrogen, albumin, serum and middle ear anti-EBV VCA IgM levels (p > 0.05). An importantfinding was that there were significant differences between groups in terms of RDW and mean platelet volume both of which may indicate chronic active inflammation (Table 1). Besides, serum anti-EBV VCA IgG levels were higher in group 2 than group 1.
The correlations between anti-EBV VCA IgG levels in serum and ear effusion samples and adenoid hypertrophy and middle ear pressure measurements are summarized inTable 2. There was also a correlation
between anti-EBV VCA IgG levels in serum and middle ear effusions (ρ value: 0.48; p value: 0.03).
ROC curve analysis resulted in a cut-off point of 2.92 S/CO level for anti-EBV VCA IgG antibodies for need for surgery in EBV infected pa-tients with 97% sensitivity and 98% specificity. Cut-off point of 17.9 S/ CO was selected for differentiation of group 1 (adenoidectomy) and group 2 (adenoidectomy + ventilation tube insertion), with 80% sen-sitivity and 80% specificity (Figs. 1 and 2).
4. Discussion
In this study, among patients with past EBV infection proved by anti-EBV VCA IgG positivity, the mainfinding was the markedly ele-vated levels of anti-EBV VCA IgG but not anti-EBV VCA IgM antibodies in both middle ear and serum samples in COME and only in serum samples in severe AH. Besides, anti-EBV VCA IgG antibody levels were further increased in COME. In previous studies, pathogens of infections and immunologic responses causing chronic inflammation were
Table 1
Blood serum values of study and control groups.
Group 1 (A) Group 2 (A + VT) Control group p value p value (post-hoc comparisons)
Median Min - Max Median Min - Max Median Min - Max
Hemoglobin (g/dL) 13.5 12–14.8 13.8 12.7–14.8 13.6 12–15.5 0.4 *0.7
**0.8 ***0.4
White Blood Cell (103/μl) 8 4.9–10 7.7 5.8–9.7 7.7 4.5–9.6 0.5 *0.9
**0.5 ***0.9 C-reactive protein (mg/dL) 0.7 0.06–7.5 0.75 0.1–4.6 0.4 0.01–4.5 0.71 *0.5 **0.06 ***0.9 Aspartate Transaminase (g/dL) 26 12–44 27 18–49 27.4 18–49 0.7 *0.9 **0.6 ***0.9 Alanine Transaminase (g/dL) 14 8–42 14.5 7–39 14 8–47 0.8 *0.9 **0.8 ***0.9
Mean Platelet Volume (fL) 7.5 4.7–12.3 6.2 4.6–8.5 8.4 5.4–12 < 0.001 *0.06
** < 0.001 *** < 0.001
RDW (%) 15.9 10.9–40.6 12.6 10.8–17.2 34 10.8–50 < 0.001 *0.66
**0.26 *** < 0.001
Serum EBV IgM (S/CO) 0.07 0–0.97 0.05 0.01–2.58 0.07 0–0.43 0.08 *0.74
**0.31 ***0.13
Serum EBV IgG (S/CO) 15.9 0.01–48 36.4 0.02–67 2.01 0.02–3.5 < 0.001 *0.66
** < 0.001 *** < 0.001
(RDW: Red cell distribution width, EBV: Ebstein-Barr virus). *: Group 1 vs. group 2.
**: Group 1 vs. control group. ***: Group 2 vs. control group.
(p < 0.05 is statistically significant and for post-hoc comparisons p < 0.017 was significant).
Table 2
The correlations between EBV IgG levels in serum and glue ear and adenoid hypertrophy and middle ear pressure measurements.
Ear Pressure Degree of Adenoid Hypertrophy Serum EBV IgG levels ρ value − 0.11 0.67
p value 0.14 < 0.001 Middle ear effusion EBV
IgG levels
ρ value − 0.53 0.27
p value 0.016 0.25
(p < 0.05 is statistically significant, ρ value is the rho value showing the cor-relation coefficient).
evaluated in COME [9,10], but current article is thefirst article which establishes that exaggerated chronic immune response to EBV (anti-EBV VCA IgG) may play a significant role in the pathogenesis of com-plications developing after acute EBV infections such as AH and COME. Another importantfinding of this study is that markedly increased serum anti-EBV VCA IgG levels may suggest a need for surgery need in
AH and COME. In this study, a cut-off point of 2.92 S/CO was found with 97% sensitivity and 98% specificity for serum anti-EBV VCA IgG levels to determine the need for surgery in both AH and COME patients. Besides another cut-off point of 17.9 S/CO was found to differentiate severe AH from COME with 80% sensitivity and 80% specificity. Thus, we can say that an easy blood sampling in AH and COME may predict
Fig. 1. ROC curve analysis of need for surgery due to severe AH and/or COME.
*ROC curve analysis resulted in a cut-off point of 2.92 S/CO level for anti-EBV IgG antibodies for need for surgery in EBV infected patients with 97% sensitivity and 98% specificity.
Fig. 2. ROC curve analysis for differentiation between severe AH and COME.
the severity and need for surgery in these patients with an acceptable sensitivity and specificity.
AH can lead to obstruction at the nasopharyngeal ostium of the Eustachian tube, resulting in negative pressure and mucosal transuda-tion in the middle ear cavity. Besides this, AH is also a medium for pathogenic microbiota (viruses, bacteria, and biofilms) and this hall-mark is the major reason for adenoidectomy in COME [5]. Despite some studies have supported the pathogenic microbiota in adenoid tissue show similarity with microbiota in middle ear [10], no studies have thoroughly studied directly anti-EBV VCA IgG antibodies showing the immune response to pathogens in the middle ear. In this study, mark-edly elevated anti-EBV VCA IgG antibodies were found not only in the serum samples but also in the effusion samples collected from the middle ear. An exaggerated immune response to EBV infection in the long term after the acute infection may explain a causal relationship between these antibodies and severity of the chronic disease.
The current study also denoted the correlation between anti-EBV VCA IgG levels and AH and middle ear pressures. High levels of serum anti-EBV VCA IgG is correlated with degree of adenoid hypertrophy and high anti-EBV VCA IgG levels in middle ear effusion is correlated with negative pressure in the middle ear. Besides, serum anti-EBV VCA IgG level is correlated with anti-EBV VCA IgG level in middle ear effusion. In previous studies the association with EBV DNA and obstructive AH and EBV DNA in middle ear was demonstrated [11–13]. The current study showed, like as EBV DNA, anti-EBV VCA IgG may predict the severity of the diseases and need for surgery.
There are some limitations in our study. Firstly, EBV DNA should have been detected in adenoid tissue and middle ear to prove EBV viral load but owing to past studies supporting this relation, we didn't want to replicate this finding [14]. Secondly, the anti-EBV VCA IgG titers might have been detected after surgical intervention but new studies can be designed to support this idea.
5. Conclusion
Markedly increased serum anti-EBV VCA IgG antibodies in children who developed upper respiratory tract complications such as severe AH and COME may show the significant role of enhanced immune system reaction in the pathogenesis of these complications due to EBV infec-tion. Besides, clinicians may easily use anti-EBV VCA IgG antibodies to decide severity and need for surgery in COME and AH.
Conflicts of interest
There is no conflict of interest between authors. References
[1] R.M. Rosenfeld, J.J. Shin, S.R. Schwartz, R. Coggins, L. Gagnon, J.M. Hackell, et al., Clinical practice guideline: otitis media with effusion (Update), Otolaryngol. Head Neck Surg. (Tokyo) 154 (2016) S1–S41,http://dx.doi.org/10.1177/
0194599815623467.
[2] M.L. Casselbrant, E.M. Mandel, Epidemiology, in: R.M. Rosenfeld, C.D. Bluestone (Eds.), Evidence-based Otitis Media, second ed., BC Decker Inc, Hamilton, Canada, 2003, pp. 147–162.
[3] R.M. Rosenfeld, D. Kay, Natural history of untreated otitis media, Laryngoscope 113 (2003) 1645–1657.
[4] L.H. Nguyen, J.J. Manoukian, A. Yoskovitch, K.H. Al-Sebeih, Adenoidectomy: se-lection criteria for surgical cases of otitis media, Laryngoscope 114 (2004) 863–866. [5] L. Nistico, R. Kreft, A. Gieseke, J.M. Coticchia, A. Burrows, P. Khampang, et al.,
Adenoid reservoir for pathogenic biofilm bacteria, J. Clin. Microbiol. 49 (2011) 1411–1420,http://dx.doi.org/10.1128/JCM.00756-10.
[6] Mandell, Douglas, Bennett, Principles and practice of infectious diseases, in: J.E. Bennett, R. Dolin, M.J. Blaser (Eds.), Epstein-barr Virus, Elsevier/Saunders, Philadelphia, PA, 2015, pp. 1765–1766.
[7] S.R. Parikh, M. Coronel, J.J. Lee, S.M. Brown, Validation of a new grading system for endoscopic examination of adenoid hypertrophy, Otolaryngol. Head Neck Surg. 135 (2006) 684–687,http://dx.doi.org/10.1016/j.otohns.2006.05.003. [8] E. Onusko, Tympanometry, Am Fam. Physician 70 (9) (2004) 1713–1720. [9] S. Arshi, F. Dehghani Firouzabadi, B. Ghalehbaghi, A. Dehghani Firouzabadi,
F. Jalali, M. Shekarabi, R. Sirous, M. Dehghani Firouzabadi, Otitis media with ef-fusion in children and the impact of risk factors on serum CytokineLevels, Iran J Otorhinolaryngol 29 (91) (2017) 75–81.
[10] G.P. Buzatto, E. Tamashiro, J.L. Proenca-Modena, T.H. Saturno, M.C. Prates, T.B. Gagliardi, L.R. Carenzi, E.T. Massuda, M.A. Hyppolito, F.C. Valera, E. Arruda, W.T. Anselmo-Lima, The pathogens profile in children with otitis media with ef-fusion and adenoid hypertrophy, PLoS One 12 (2) (2017) e0171049,http://dx.doi. org/10.1371/journal.pone.0171049.
[11] H. Faden, V. Callanan, M. Pizzuto, M. Nagy, M. Wilby, D. Lamson, B. Wrotniak, S. Juretschko, K. St George, The ubiquity of asymptomatic respiratory viral infec-tions in the tonsils and adenoids of children and their impact on airway obstruction, Int. J. Pediatr. Otorhinolaryngol. 90 (2016) 128–132,http://dx.doi.org/10.1016/j. ijporl.2016.09.006.
[12] S. Al-Salam, S.A. Dhaheri, A. Awwad, S. Daoud, A. Shams, M.A. Ashari, Prevalence of Epstein-Barr virus in tonsils and adenoids of United Arab Emirates nationals, Int. J. Pediatr. Otorhinolaryngol. 75 (9) (2011) 1160–1166,http://dx.doi.org/10.1016/ j.ijporl.2011.06.012.
[13] X.C. Xue, X.P. Chen, W.H. Yao, Y. Zhang, G.B. Sun, X.J. Tan, Prevalence of human papillomavirus and Epstein-Barr virus DNA in Chinese children with tonsillar and/ or adenoidal hypertrophy, J. Med. Virol. 86 (6) (2014) 963–967,http://dx.doi.org/ 10.1002/jmv.23894.
[14] C. Besson, C. Amiel, C. Le-Pendeven, P. Brice, C. Fermé, P. Carde, O. Hermine, M. Raphael, L. Abel, J.C. Nicolas, Positive correlation between Epstein-Barr virus viral load and anti-viral capsid immunoglobulin G titers determined for Hodgkin's lymphoma patients and their relatives, J. Clin. Microbiol. 44 (1) (2006) 47–50,
