Keywords
viruses - adenoids - adenoidectomy - child - polymerase chain reaction
Introduction
The Waldeyer lymphatic ring is formed by a conglomerate of lymphoid structures comprising
the pharyngeal tonsils (adenoids) and palatine tonsils, beyond the lingual and peritubal
tonsils. It is strategically located at the entrance of the respiratory and digestive
systems and is exposed to a wide variety of antigens present in air and food, which
are continuously presented to the body.[1]
[2]
[3]
Hypertrophy of pharyngeal and palatine tonsils is one of the most common disorders
found by otolaryngologists and is a major cause of upper airway obstruction, accounting
for ∼75% of respiratory disorders in childhood, which may vary from primary snoring
to severe cases of obstructive sleep apnea with cardiovascular commitment.[4]
[5]
The pathogenesis of inflammatory/infectious disease of tonsils and adenoids probably
involves their anatomical location and its antigen-processing function. There is no
certainty about what determines the onset of chronic infection. Viral infection with
bacterial infection may be one of the triggering mechanisms of chronic infection,
but the effects of the environment, personal factors, and diet, among others, may
also be involved. Several studies and theories have been published based primarily
on allergic mechanisms or viral and/or bacterial infectious processes.[6]
[7]
[8]
[9]
Although it is a very common health problem in daily medical practice, the presence
of respiratory viruses in lymphoid tissues of the nasopharynx and oropharynx and their
impact on recurrent infections and hypertrophy of these tissues are not yet fully
understood and are being studied by numerous researches. Many authors have identified
the presence of multiple viral agents in these tissues, latent in asymptomatic patients.[6]
[10]
[11]
[12]
[13]
[14]
[15] Most of these studies in the literature are performed among symptomatic and hospitalized
children with acute disease, which hampers a better understanding of this pathophysiology.
Aim
This study aims to identify and determine the prevalence of major respiratory viruses
in nasopharyngeal secretions collected pre- and postoperatively and adenoid tissue
of children undergoing adenoidectomy.
Methods
A prospective observational study was performed in 36 patients under 12 years of age
with upper airway lymphoid hypertrophy undergoing adenoidectomy or adenotonsillectomy,
assisted by the otorhinolaryngology clinic of a university hospital. The study was
approved by the institutional research ethics committee (protocol number 363/2011).
All parents or guardians signed a consent form after being informed of the objectives,
procedures, and responsibilities of the research, as well as received answers to any
questions regarding the study.
The study included children under 12 years of age with clinical and/or radiologic
criteria for adenoidectomy or adenotonsillectomy (recurrent tonsillitis and/or hypertrophy
of lymphoid tissues leading to upper airway obstruction). Children were excluded from
the study if they had fever or any type of acute respiratory tract infection at the
time of collection of biological samples, if they had craniofacial malformations,
if they were immunocompromised, or if their parents did not agree with their participation
in the research project.
Several respiratory viruses (influenza A and B; parainfluenza 1, 2, 3, and 4; rhinovirus;
respiratory syncytial virus; human bocavirus; coronaviruses; and metapneumovirus)
were investigated by quantitative real-time polymerase chain reaction (q-PCR) in adenoid
tissue removed surgically and nasal swab specimens collected preoperatively and at
1 month postoperative follow-up visit.
Nasal secretion was collected with the aid of a sterile metal rod nasal swab, following
the rules of asepsis. The nasal cavity was previously humidified with 1 mL of sterile
sodium chloride 0.9% solution. The swab was introduced directly into the nasal cavity
without contact with the patient's skin or other region. After collecting, this swab
was homogenized in microtubes containing sterile Ringer lactate solution. The adenoid
tissue was obtained by cold adenoidectomy under general anesthesia by curettage of
adenoid tissue. Small fragments of adenoid tissue were stored in microtubes containing
virus transport media, which is a solution for transportation and storage of viruses
in tissues.
After collection, the samples were transported in liquid nitrogen in microtubes to
the Research Laboratory of Virology and Molecular Biology, where they were frozen
at −80°C, and subsequently viral genetic material in the secretion and adenoid tissue
was extracted according to a standardized protocol by the laboratory. After extraction,
q-PCR was performed of respiratory viruses using the handset Applied Biosystems 7500
Real-Time PCR Systems (Life Technologies™, California, USA) following the manufacturer's
specifications and protocols. The FTD-Respiratory-21 kit from Fast-Track Diagnostics™
(Junglinster, Luxemburg) was used to identify respiratory viruses.
The database was created using Microsoft Excel program (Microsoft, Redmond, Washington,
United States). The data were analyzed using absolute (n) and relative (%) frequencies, average, and standard deviation, depending on the
variable studied. The averages of continuous outcome variables were compared by the
Student t test. The association of the qualitative measures between groups was performed using
the chi-square test or Fisher exact test to determine statistical significance. The
software used for analysis was SPSS (Statistical Package for Social Sciences, IBM™,
Armonk, New York, USA) version 13 and the assumed level of significance was 5% (p < 0.05).
Results
The study evaluated 36 children between 3 and 12 years of age undergoing adenoidectomy
or adenotonsillectomy from April 2012 to January 2013, with an average age of 7.3
years and a median of 7 years. Of these 36 children, 25 (69.4%) were girls and 11
(30.6%) were boys. A total of 108 pre- and postoperative nasopharyngeal specimens
and adenoid tissue samples were removed surgically from these 36 patients (75 samples
from girls and 33 from boys). At least 1 viral agent was isolated in any of the samples
collected in 58.3% of 36 children (n = 21) and in 25.9% of 108 samples (n = 28), in 36.4% (n = 12) of 33 samples of boys and 21.3% (n = 16) samples from 75 girls. Of the respiratory viruses found in 36 samples, 75%
(n = 27) were isolated in children under 7 years of age.
Of the 21 patients with positive results for any respiratory virus, 6 (28.6%) had
more than 1 respiratory virus. Considering the 28 positive samples, the rate of viral
coinfection also was 28.6% (n = 8): 3 samples of adenoid tissue (27.3%), 2 samples of preoperative secretion (16.7%),
and 3 samples of postoperative secretion (60%). All coinfections were observed in
patients under 6 years of age, and 2 of these patients had coinfections in 2 different
samples.
Respiratory viruses were found in 33.3% of preoperative nasopharyngeal specimens (n = 12), with a single virus isolated in 83.3% (n = 10) and coinfection by 2 viral agents in 16.7% of samples (n = 2), totaling 14 respiratory viruses. In adenoid tissue samples, respiratory viruses
were found in 30.6% (n = 11): single viral infection in 72.8% (n = 8) and coinfection in 27.2% (n = 3) of these positive samples (totaling 14 viruses). Of postoperative nasopharyngeal
secretions, the positivity was less—in only 13.9% of samples (n = 5) was a respiratory virus isolated: coinfection in 60% (n = 3) and single virus infection in 40% (n = 2; [Table 1]).
Table 1
Respiratory viruses (n = 36) found in 28 samples from 21 patients
|
Patient
|
Sex
|
Age
|
Respiratory viruses
|
|
Preoperative secretion
|
Adenoid tissue
|
Postoperative secretion
|
|
1
|
Male
|
11
|
Rhinovirus
|
–
|
–
|
|
2
|
Female
|
5
|
–
|
Parechovirus + Bocavirus
|
Parechovirus + Coronavirus 229
|
|
3
|
Male
|
5
|
Rhinovirus
|
–
|
Rhinovirus + Adenovirus
|
|
4
|
Male
|
8
|
–
|
Enterovirus
|
Adenovirus
|
|
5
|
Male
|
4
|
Bocavirus
|
–
|
–
|
|
6
|
Male
|
5
|
Rhinovirus
|
Adenovirus
|
–
|
|
7
|
Male
|
9
|
Rhinovirus
|
–
|
–
|
|
8
|
Female
|
10
|
–
|
–
|
Coronavirus HKU
|
|
9
|
Female
|
6
|
Rhinovirus + Influenza A
|
–
|
–
|
|
10
|
Female
|
6
|
Enterovirus
|
–
|
–
|
|
11
|
Female
|
10
|
–
|
Enterovirus
|
–
|
|
12
|
Female
|
4
|
Rhinovirus + Bocavirus
|
Parechovirus
|
Metapneumovirus A/B + Bocavirus
|
|
13
|
Female
|
3
|
–
|
Coronavirus 43
|
–
|
|
14
|
Male
|
5
|
–
|
Rhinovirus
|
–
|
|
15
|
Female
|
9
|
Coronavirus HKU
|
–
|
–
|
|
16
|
Male
|
7
|
Parainfluenza 4
|
–
|
–
|
|
17
|
Female
|
4
|
Rhinovirus
|
–
|
–
|
|
18
|
Female
|
5
|
–
|
Rhinovirus + Bocavirus
|
–
|
|
19
|
Female
|
9
|
–
|
Adenovirus
|
–
|
|
20
|
Male
|
5
|
–
|
Bocavirus
|
–
|
|
21
|
Female
|
6
|
Bocavirus
|
Parainfluenza 4 + Bocavirus
|
–
|
From the 14 respiratory viruses identified in preoperative nasopharyngeal secretions,
50% were rhinovirus (n = 7), present in 19% of 36 samples tested. In adenoid tissue, respiratory viruses
were identified in 14, and the most frequent virus found was bocavirus, present in
11% of samples (n = 4), corresponding to 28.6% of the virus isolated. In nasopharyngeal secretions
collected after surgery, only 8 respiratory viruses were identified, of which 25%
(n = 2) were adenoviruses, present in 6% of samples.
Considering all 36 respiratory viruses isolated in 108 samples evaluated, the main
agent isolated was rhinovirus (27.8%), followed by bocavirus (22.2%). Other viruses
isolated with a lower frequency were adenovirus (11.1%), enteroviruses (8.3%), parechovirus
(8.3%), coronavirus HKU (5.6%), parainfluenza virus 4 (5.6%), influenza A (2.8%),
coronavirus 43 (2.8%), coronavirus 229 (2.8%), and metapneumovirus A/B (2.8%). The
respiratory syncytial virus was not isolated in the samples ([Table 2]).
Table 2
Frequency of viruses in the samples (n = 36)
|
Preoperative secretion
|
Adenoid tissue
|
Postoperative secretion
|
Total
|
|
Rhinovirus
|
7 (50%)
|
2 (14.3%)
|
1 (12.5%)
|
10 (27.7%)
|
|
Bocavirus
|
3 (21.4%)
|
4 (28.6%)
|
1 (12.5%)
|
8 (22.2%)
|
|
Adenovirus
|
0 (0%)
|
2 (14.3%)
|
2 (25%)
|
4 (11.1%)
|
|
Enterovirus
|
1 (7.2%)
|
2 (14.3%)
|
0 (0%)
|
3 (8.3%)
|
|
Parechovirus
|
0 (0%)
|
2 (14.3%)
|
1 (12.5%)
|
3 (8.3%)
|
|
Coronavirus HKU
|
1 (7.2%)
|
0 (0%)
|
1 (12.5%)
|
2 (5.6%)
|
|
Parainfluenza 4
|
1 (7.2%)
|
1 (7.1%)
|
0 (0%)
|
2 (5.6%)
|
|
Influenza A
|
1 (7.2%)
|
0 (0%)
|
0 (0%)
|
1 (2.8%)
|
|
Coronavirus 43
|
0 (0%)
|
1 (7.1%)
|
0 (0%)
|
1 (2.8%)
|
|
Coronavirus 229
|
0 (0%)
|
0 (0%)
|
1 (12.5%)
|
1 (2.8%)
|
|
Metapneumovirus A/B
|
0%
|
0 (0%)
|
1 (12.5%)
|
1 (2.8%)
|
|
Total
|
14 (100%)
|
14 (100%)
|
8 (100%)
|
36 (100%)
|
Discussion
In general, 58.4% of the patients (n = 21) and 25.9% of samples (n = 28) were positive for at least 1 respiratory virus. Studies in the literature show
different prevalences, because some studies were virus-specific and others researched
the prevalence of multiple respiratory viruses at the same time. Herberhold et al
investigated several respiratory viruses in adenoid tissue from 30 children. The authors
found a high positivity: at least 1 respiratory virus was identified in 97% of 52
samples.[16] Sato et al also searched respiratory viruses in adenoid tissue and found at least
1 respiratory virus in all samples.[17] In the study of Proenca-Modena et al, this positivity was 97.5%, and the highest
rate of viral detection was found in the adenoid tissue (85.7%), followed by nasal
secretions (78.5%), tonsils (68.6%), and peripheral blood (1%).[8]
Considering all viruses found in this study, rhinovirus was isolated most frequently,
followed by bocavirus. In the study of Herberhold et al, rhinovirus was also the most
common respiratory virus, identified in 67% of samples, followed by bocavirus in 53%,
similar to our study but with different prevalences.[16] In another study, adenovirus was the most common viral agent, isolated in 47.1%
of patients, followed by enterovirus (40.5%), rhinovirus (38%), and bocavirus (29.8%).[8] In the study from Sato et al, adenovirus was the most common viral agent, present
in 80% of samples.[17] Alkhalaf et al analyzed 106 palatine and pharyngeal tonsils of 57 patients undergoing
routine tonsillectomy or adenoidectomy. Employing the technique of real-time polymerase
chain reaction, the authors identified 84 samples (72.4%) positive for adenovirus.[18]
In this study, 28.6% (n = 6) of patients with positive results presented coinfection with 2 viral agents,
and 2 patients had viral coinfection in 2 samples, totaling 8 samples. Bocavirus was
isolated in 75% (n = 6) of the coinfections. Rhinovirus was identified in all coinfections of preoperative
nasopharyngeal secretions (n = 2), and bocavirus was isolated in all coinfections in adenoid tissue (n = 3). In coinfections of preoperative nasopharyngeal secretions, there was no predominant
viral agent.
Herberhold et al found multiple respiratory viruses in 83% of samples evaluated.[16] In the study of Proenca-Modena et al, the rate of coinfection was 69.5%. The authors
evaluated 121 children who underwent adenotonsillectomy and found high rates of viral
detection in adenotonsillar tissues. The authors were researching respiratory viruses
on palatine tonsils, adenoid tissue, nasal secretions, and peripheral blood but did
not evaluate the postoperative nasopharyngeal secretions.[8]
Review of the literature found no studies comparing the prevalence of the virus in
nasopharyngeal secretions pre- and postoperatively. Some studies revealed that the
adenoid tissue may be a reservoir of viruses. Comparing the prevalence of virus pre-
and postoperatively, we aimed to analyze a possible influence of adenoid tissue in
the maintenance of these viruses in secretions. After the analysis of preoperative
nasopharyngeal samples, this study found that 33.3% of patients (n = 12) were positive for at least 1 respiratory virus. Of the 14 respiratory viruses
isolated, 50% were rhinovirus. After surgery, the positivity of respiratory viruses
in nasopharyngeal secretions of these patients decreased: in only 13.9% of patients
(n = 5) was at least 1 viral agent isolated in the sample. In the postoperative secretions,
8 respiratory viruses were detected, and adenoviruses were identified in 2 patients.
These results suggest a decrease in the prevalence of the virus after surgery (p = 0.0522). However, the number of patients in the study is relatively small, and
a greater number of samples are required to confirm or reject this hypothesis.
In addition to the small sample size, a seasonal effect may contribute to the discrepancies
found between our results and other studies in the literature. We know that some respiratory
viruses are found in certain periods of the year; however, as we have seen, apparently
the virus may persist in the adenoid tissue and nasopharyngeal secretions even after
the symptomatic phase of the disease. The climatic conditions of each region, air
quality, and population characteristics may also contribute to a greater or lesser
circulation of respiratory viruses in a given region and time of year.
The present study revealed that some patients with hypertrophy of pharyngeal tonsil
exhibit respiratory viruses detected on nasopharyngeal and adenoid tissue, even if
asymptomatic. These data suggest that the persistence or latency of respiratory viruses
on nasopharyngeal and adenoid tissue after an acute infectious process may be related
to the pathogenesis of lymphoid hypertrophy of the upper airways and that these tissues
may function as a reservoir of virus, with possible influence on its transmission
in the community. After adenoidectomy, a decrease in respiratory viruses present in
nasopharyngeal secretions was observed. From this finding, we can infer a lower chance
of occurrence of infections as well as secondary superinfections and virus transmission
to contacts after surgery. Considering the age group studied, in which there is a
greater interpersonal contact, this decrease in the circulation of respiratory viruses
becomes important for public health, with positive impact on the quality of life of
these children.
Conclusion
The rhinovirus was the most frequently found virus in all samples, followed by bocavirus.
After removal of adenoid tissue, there was a decrease in the prevalence of viruses
in nasopharyngeal secretions.
