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Acta Oto-Laryngologica, 2010; 130: 620625

ORIGINAL ARTICLE

Atypical bacteria in adenoids and tonsils of children requiringadenotonsillectomy

GIORGIO L. PIACENTINI1, DIEGO G. PERONI1, FRANCESCO BLASI2,

LYDIA PESCOLLDERUNGG3, PAUL GOLLER4, LORENZ GALLMETZER5,

LORENZO DRAGO6, ALESSANDRO BODINI1 & ATTILIO L. BONER1

1Clinica Pediatrica, Universit di Verona, Verona,

2Institute of Respiratory Diseases, University of Milan,

IRCCS Fondazione POMARE, Milan,3

Divisione di Pediatria, Ospedale Regionale, Bolzano,4

Divisione ORL,

Ospedale Bressanone, Bolzano,5

Divisione ORL, Ospedale Regionale, Bolzano and6

Lab of Clinical Microbiology,

Department of Preclinical Science LITA, University of Milan, Italy

Abstract

Conclusions: The results of this study suggest that atypical bacteria may be involved not only in acute upper airway diseases but

also in recurrent infections requiring adenoidectomy and/or tonsillectomy. Therefore, their identification, followed by an

appropriate treatment, should be considered. Objective: Although viruses and group A beta-haemolytic streptococci (GABHS)

represent the most frequent bacterial aetiological agents of paediatric upper respiratory tract infections (URTIs), chlamydia

and Mycoplasma pneumoniae have also been found in acute tonsillopharyngitis. Nevertheless their relevance in chronic or

recurrent URTI has never been evaluated. This study aimed to further address the role of atypical bacteria in recurrent URTIs

requiring adenoidectomy and tonsillectomy. Methods: Samples from 55 consecutive children who underwent adenoidectomy

and/or tonsillectomy for recurrent or chronic URTI were cut transversely into smaller sections of 5 mm. Each section was

pooled and assayed by specific PCR for viruses and bacteria. Results: Adenovirus was detected in 10 patients (18.2%),

influenza A virus in one patient and influenza B virus in another. None of the other tested viruses was found. GABHS was

found in 37 patients (67.3%). Moraxella catarrhalis and Haemophilus influenzae were detected in 30 patients (54.5%).

M. pneumoniae was detected in 6 patients (10.9%) and C. pneumoniae was found in 10 patients (18.2%).

Keywords: URTI, PCR, virus

Introduction

Respiratory viruses are the major cause of upper

respiratory tract infections (URTIs). Bacteria account

for 530% of all pharyngitis episodes. Group A beta-

haemolytic Streptococcus (GABHS) is the most com-

monly isolated bacterium in throat culture. Manyother bacteria such as Staphylococcus aureus, Strep-

tococcus pneumoniae, Haemophilus influenzae,

Moraxella catarrhalis, Haemophilus parainfluenzae

and anaerobic bacteria have been isolated from sur-

face or core tonsillar cultures [1]. Komaroff and

co-workers [2] have demonstrated that Chlamydia

pneumoniae and Mycoplasma pneumoniae have

been found in adults with acute tonsillopharyngitis.

Some of these studies [2,3] have demonstrated that

atypical bacteria can be found in samples from

patients with URTIs, therefore suggesting that these

organisms should be considered in the differential

diagnosis and in the therapeutic choices for pharyn-gotonsillitis. Nevertheless, the only few previous stud-

ies performed in the 1990s in tonsil tissues were not

conclusive in addressing the role of atypical bacteria,

chlamydia and Mycoplasma pneumoniae, in recur-

rent or chronic tonsillitis and pharyngitis [4,5]. In

fact, while a study by Charnock and co-workers [4]

Correspondence: Prof. Giorgio Piacentini, Clinica Pediatrica, Policlinico G.B. Rossi, 37134 Verona, Italy. Tel: +39 (0)45 8200993. Fax: +39 (0)45 8124744.

E-mail: giorgio.piacentini@univr.it

(Received 8 May 2009; accepted 20 July 2009)

ISSN 0001-6489 print/ISSN 1651-2251 online 2010 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)

DOI: 10.3109/00016480903359921

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suggested that chlamydia and mycoplasma were not

involved in chronic tonsillitis, studies by Falck and

co-workers [5] have demonstrated that C. pneumo-

niae was the agent of chronic pharyngitis and that it

was found in tissue from the retropharyngeal mucosal

membrane [5]. The aim of our study was to furtheraddress the issue of the role of atypical bacteria in

recurrent URTI requiring adenoidectomy and

tonsillectomy.

Material and methods

Samples from 55 consecutive patients, ranging in age

from2to16years(medianage5years)(Q1 =4;Q3=7),

who underwent adenoidectomy and/or tonsillectomy

for recurrent or chronic URTI (defined as seven docu-

mented upper airways infections with clinical signs of

adenoiditis and/or tonsillitis accompanied by sorethroat and fever in the previous year), were evaluated.

Patients who had received antibiotic treatment in the

previous week were not admitted to the study. Patients

receiving benzathine penicillin G in the previous

4 weeks were also excluded. Eight of the patients

underwent both adenoidectomy and tonsillectomy.

Immediately after removal, tissues were aseptically

cut in aliquots that were then used for microbiological

assays and frozen. Samples were stored at 80C until

nucleic acid extraction.

The Ethical Committee of the Hospital of Bolzano

approved the study design. The patients or the parents

of young children gave their consent to participate tothe study.

Viral detection

The molecular methods for detection of respiratory

viruses included four real-time specific PCR assays

(Fast set, Arrows Diagnostics Srl, Genoa, Italy) for

the following: respiratory syncytial virus (RSV) A

and B, influenza virus A and B, parainfluenza virus

1, 2 and 3, and adenoviruses.

Nucleic acids were extracted using the RNeasy

Mini Kit (Qiagen, Milan, Italy) according to the

protocol for isolation of nucleic acids from animal

tissues. After tissue homogenation in 600 ml buffer,

each sample was eluted in 100 ml RNase-free water,

which was sufficient for all real-time analyses.

Real-time PCR was carried out with a Rotor-Gene

3000 (Corbett Research, Cambridge,UK) set in accor-

dancewiththeFastsetprotocol.Primersweredesigned

from conserved regions of genes codifying the Matrix

Protein, the Nucleoprotein, the Fusion protein, hae-

magglutininneuroaminidase, and the hexon antigen of

influenza virus type A, B and RSV, parainfluenzae

viruses and adenovirus, respectively. For RSV detec-

tion, the reaction mixture consisted of 2 reaction mix

(Invitrogen, Carlsbad, CA, USA), PCR additive I,

RSV mix (primer 300 nM, TaqMan probe 100 nM),

SuperScript III RT/Platinum Taq mix, RNase OUTand 10ml of template, with a total volume of 50ml. The

PCRthermalprofileconsistedofaninitialRTstepof15

min at50C followedby 2 min at95C and 35cycles of

1 5 sa t 9 5Cand60sat60C. RSV signal was acquired

on FAM channel (excitation wavelength470 nm, emis-

sion wavelength 510 nm).

With regard to influenza type A and B, 10 ml of

template were added to the reaction mixture consist-

ing of 2 reaction mix (Invitrogen), PCR additive I,

Flu mix (primer 250 nM, TaqMan probe 75 nM),

SuperScript III RT/Platinum Taq mix, RNase OUT,

with a total volume of 50 ml. The PCR thermal profile

consisted of an initial RT step of 15 min at 50

Cfollowed by 2 min at 95C and 35 cycles of 15 s at

95C and 60 s at 62C. Multiple fluorescent signals

were obtained with detectors corresponding to FAM

(excitation wavelength 470 nm, mission wavelength

510 nm) and JOE (excitation wavelength 530 nm,

emission wavelength 555 nm) channels, respectively.

For parainfluenzae virus detection, 14 ml of template

were added to the reaction mixture consisting of 2

reaction mix (Invitrogen), 50 mM MgSO4 (Invitro-

gen) PIV mix (primer 250 nM, TaqMan probe

75 nM), SuperScript III RT/Platinum Taq mix, RNase

OUT, with a total volume of 50 ml. The PCR thermal

profileconsisted of aninitial RT step of 15 min at 50Cfollowed by 2 min at 95C and 40 cycles of 15 s at

95C, 20 s at 57C and 15 s at 72C. Parainfluenzae

virus signal was acquired on FAM channel (excitation

wavelength 470 nm, emission wavelength 510 nm).

For adenovirus detection, the reaction mixture con-

sisted of water, 50 mM MgCl2, Adeno_R mix (primer

300 nM, TaqMan probe 100 nM), Premix Ex Taq 2

and 15ml of template, with a total volume of 50ml. The

PCR thermal profile consisted of an initial step at 95C

for 30 s followed by 35 cycles of 15 s at 95 C, 30 s at

58C and 10 s at 72C. Amplification signal was

acquired on FAM channel (excitation wavelength

470 nm, emission wavelength 510 nm).

A threshold cycle (Ct) value for each sample was

calculated, determining the point at which the fluo-

rescence exceeded a threshold limit of 0.01.

Bacterial detection

Each tonsil was cut transversely into smaller sections

of 5 mm. Each section was pooled and assayed by

specific PCR.

Paediatric adenotonsils requiring adenotonsillectomy 621

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Chromosomal DNA was extracted by a commercial

kit (Roche Diagnostics, Germany).

As regards PCR amplification, to confirm the extrac-

tion each DNA sample was tested for its ability to be

amplified with B-globin specific primers [6]. For the

detection ofC.pneumoniae bynested PCR,primersthatamplify the 207 bp fragment of the major outer mem-

brane proteingenes (ompA) wereused [7].Primers that

amplify 104 bp fragment P1 protein antigen [8] were

used for the detection of Mycoplasma pneumoniae by

nested PCR. For the detection of H. influenzae, Mor-

axella catarrhalis and Streptococcus pneumoniae, the

bacterial 16S rRNA gene was chosen as target and one

common lower primer and three species-specific upper

primers were used in a multiplex PCR [9]. Primers that

amplify 504 bp fragment oprL gene were used for the

detectionofPseudomonasaeruginosa[10].Primersthat

amplify M protein gene were used for the detection of

GABHS [11]. For Legionella pneumophila the ampli-fied gene in nested PCR was macrophage infectivity

potentiator protein (MIP) [12].

After amplification, 4% agarose gel electrophoresis

and ethidium bromide staining were used to visualize

the PCR products.

Statistical analysis

Categorical data were analysed using contingency

analysis and a c2 or a Fishers test.

Results

Adenovirus was detected in 10 patients (18.2%): 3 in

the tonsils (16.7% of the samples) and 7 in the

adenoids (25.9% of the samples) (NS). Influenza A

was detected in the adenoids of one patient and

influenza B in the tonsils of another. None of the

other tested viruses was found. The percentages of

positive results for the investigated bacteria for ton-

sillar and adenoid findings are reported in Figure 1.

GABHS was found in 37 patients (67.3%); it was

found in both the adenoids and the tonsils in 5 of the

8 patients (62.5%) who had both adenoids and tonsils

removed, whereas only 1 of these 8 patients was

GABHS-negative in both the specimens. GABHS

was detected in 21 of the 33 adenoid samples

(66.6%), and in 16 of 30 tonsil samples (53.3%).

Moraxella catarrhalis and H. influenzae were detected

in 30 patients (54.5%); these bacteria were detected in20 of the 33 adenoid samples (60.6%) and in 14 of the

30 tonsil samples (46.7%) (NS). Moraxella catarrhalis

and H. influenzae were found in both adenoids and

tonsils in four of the eight patients who underwent the

dual surgery.

Mycoplasma pneumoniae was detected in 6 of

55 patients (10.9%); in 4 patients it was found in

the adenoids and in 2 in the tonsils and it was not

detected in both tissue specimens in any of the patients.

C. pneumoniae was found in 10 patients (18.2%);

in 5 patients in the adenoids and in 5 subjects in the

Tonsils (n=22)

Adenoids (n=25)

70

60

50

40

30

20

10

0

GABHS

M.catarrhalis

H.

influenzae

C.pneumoniae

M.pneumoniae

Adenoids + tonsils (n=8)

Figure 1. Percentages of positive results for the investigated bacteria in tonsillar and adenoid findings. GABHS, group A beta-hemolytic

Streptococcus.

622 G.L. Piacentini et al.

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tonsils. It was not detected in both the adenoids and

tonsils in any of the patients. No patients showed the

concomitant presence of the two atypical bacteria,

therefore 16 patients (29.1%) harboured either one or

the other atypical bacteria.

S. pneumoniae was detected in 4 of 22 (18%) of the

patients who underwent tonsillectomy, 4 of 25 (26%)

of those with adenoidectomy and in 2 of the 8 (25%)

subjects who underwent dual surgery.L. pneumophila was never found.

Eighteen patients (32.7%) carried both GABHSand

Moraxella catarrhalis or H. influenzae and in

11 patients (20%) both GABHS and Mycoplasma

pneumoniae or C. pneumoniae were detected.

GABHS was significantly more frequently found

than viruses (p

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organisms can be found in such patients more often

than had been suspected [2]. Furthermore, Falck and

co-workers [5] suggested a potential implication of

atypical bacteria not only in acute but also in the case

of chronic pharyngitis. More recently, Esposito and

co-workers [3,4] demonstrated that atypical bacteriacan be found in children with acute and recurrent

pharyngitis and tonsillopharyngitis. The potential role

of C. pneumoniae and Mycoplasma pneumoniae in

recurrent or chronic URTI is of primary relevance,

not only from an epidemiological consideration, but

mainly because of the response of these bacteria to

macrolides rather than to other classes of antibiotics.

In our study, around 10% of the patients were PCR-

positive for Mycoplasma pneumoniae and 20% for C.

pneumoniae. As a consequence, approximately 30%

of our patients showed the presence of genetic mate-

rial from atypical pathogens in adenoids and tonsils,

which represents a clinically significant proportion ofpatients and it gives support to the results of the

previous study showing the presence of atypical bac-

teria in patients with recurrent infection of the upper

airway [5]. Our prevalence rate for Mycoplasma

pneumoniae and C. pneumoniae infection is exactly

the same as that previously documented, with a four-

fold increase in specific antibodies, in 763 adult

patients presenting with pharyngitis [2] and similar

to that found by means of PCR for C. pneumoniae in

adults with acute pharyngolaryngitis [13]. Thefinding

of similar prevalence in adults, studied during the

acute phase of disease, and in children studied

throughout the chronic phase of illness, is not sur-prising. In fact, C. pneumoniae infection is charac-

terized by its persistence in infected tissue [14] and,

consequently, it is very likely that adult patients,

studied at an acute point of their disease, were already

infected with atypical bacteria. Persistent infection has

also been suggested for Mycoplasma pneumoniae

[14]. For C. pneumoniae, persistence naturally

occurs in monocytes/macrophages, cells highly repre-

sented in adenoids and tonsils where they might

contribute to the pathogenesis of recurrent disease.

It is possible that both Mycoplasma pneumoniae

and C. pneumoniae are merely associated with recur-

rent URTI rather than having a pathogenetic effect.

Unfortunately, the main limitations of the experimen-

tal design of this study were the lack of a control group

of children undergoing adenoidectomy and tonsillec-

tomy for airway obstruction in the absence of signif-

icant recurrent URTI and a diagnosis of aetiology

prior to surgery, which would have supported the

aetiological role of the different pathogens in the

disease. Such a limitation was due to the real-life

design of our study, which aimed to evaluate the level

of colonization by atypical bacteria in a general pop-

ulation rather than in selected groups.

In conclusion, although the data from the present

study cannot prove an aetiological role, several of the

organisms recovered in the specimens obtained from

our patients could have been the key aetiologicalagents for the recurrent episodes of infection that

suggested the opportunity for a tonsillectomy or ade-

noidectomy. Therefore, in consideration of the ten-

dency for atypical bacteria to persist at the site of

infection and to induce chronic inflammation, we

suggest consideration of their identification and

appropriate treatment in children with recurrent

upper airway illness. Further studies with a longitu-

dinal design and a control group undergoing adenoi-

dectomy and tonsillectomy for airway obstruction

rather than for recurrent infections are warranted to

investigate the aetiological role of atypical bacteria in

recurrent tonsillopharyngitis and to assess the effect ofantibiotic therapy.

Declaration of interest: The authors report no

conflicts of interest. The authors alone are responsible

for the content and writing of the paper.

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