Acta Oto-Laryngologica, 2010; 130: 620625

ORIGINAL ARTICLE

Atypical bacteria in adenoids and tonsils of children requiring adenotonsillectomy

GIORGIO L. PIACENTINI1, DIEGO G. PERONI1, FRANCESCO BLASI2, LYDIA PESCOLLDERUNGG3, PAUL GOLLER4, LORENZ GALLMETZER5, LORENZO DRAGO6, ALESSANDRO BODINI1 & ATTILIO L. BONER1
1

Clinica Pediatrica, Universit di Verona, Verona,2Institute of Respiratory Diseases, University of Milan, IRCCS Fondazione POMARE, Milan,3Divisione di Pediatria, Ospedale Regionale, Bolzano,4Divisione ORL, Ospedale Bressanone, Bolzano,5Divisione ORL, Ospedale Regionale, Bolzano and 6Lab 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 identication, 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 specic PCR for viruses and bacteria. Results: Adenovirus was detected in 10 patients (18.2%), inuenza A virus in one patient and inuenza B virus in another. None of the other tested viruses was found. GABHS was found in 37 patients (67.3%). Moraxella catarrhalis and Haemophilus inuenzae 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 betahaemolytic Streptococcus (GABHS) is the most commonly isolated bacterium in throat culture. Many other bacteria such as Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus inuenzae, Moraxella catarrhalis, Haemophilus parainuenzae and anaerobic bacteria have been isolated from surface 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 pharyngotonsillitis. Nevertheless, the only few previous studies performed in the 1990s in tonsil tissues were not conclusive in addressing the role of atypical bacteria, chlamydia and Mycoplasma pneumoniae, in recurrent 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

Paediatric adenotonsils requiring adenotonsillectomy suggested that chlamydia and mycoplasma were not involved in chronic tonsillitis, studies by Falck and co-workers [5] have demonstrated that C. pneumoniae 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 further address the issue of the role of atypical bacteria in recurrent URTI requiring adenoidectomy and tonsillectomy.

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Material and methods Samples from 55 consecutive patients, ranging in age from 2 to 16 years (median age 5 years) (Q1 = 4; Q3 = 7), who underwent adenoidectomy and/or tonsillectomy for recurrent or chronic URTI (dened as seven documented upper airways infections with clinical signs of adenoiditis and/or tonsillitis accompanied by sore throat 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 80 C 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 to the study.

Viral detection The molecular methods for detection of respiratory viruses included four real-time specic PCR assays (Fast set, Arrows Diagnostics Srl, Genoa, Italy) for the following: respiratory syncytial virus (RSV) A and B, inuenza virus A and B, parainuenza 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 sufcient for all real-time analyses. Real-time PCR was carried out with a Rotor-Gene 3000 (Corbett Research, Cambridge, UK) set in accordance with the Fast set protocol. Primers were designed from conserved regions of genes codifying the Matrix Protein, the Nucleoprotein, the Fusion protein, haemagglutinin neuroaminidase, and the hexon antigen of

inuenza virus type A, B and RSV, parainuenzae viruses and adenovirus, respectively. For RSV detection, 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 OUT and 10 ml of template, with a total volume of 50 ml. The PCR thermal prole consisted of an initial RT step of 15 min at 50 C followed by 2 min at 95 C and 35 cycles of 15 s at 95 C and 60 s at 60 C. RSV signal was acquired on FAM channel (excitation wavelength 470 nm, emission wavelength 510 nm). With regard to inuenza type A and B, 10 ml of template were added to the reaction mixture consisting 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 prole consisted of an initial RT step of 15 min at 50 C followed by 2 min at 95 C and 35 cycles of 15 s at 95 C and 60 s at 62 C. Multiple uorescent 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 parainuenzae virus detection, 14 ml of template were added to the reaction mixture consisting of 2 reaction mix (Invitrogen), 50 mM MgSO4 (Invitrogen) 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 prole consisted of an initial RT step of 15 min at 50 C followed by 2 min at 95 C and 40 cycles of 15 s at 95 C, 20 s at 57 C and 15 s at 72 C. Parainuenzae virus signal was acquired on FAM channel (excitation wavelength 470 nm, emission wavelength 510 nm). For adenovirus detection, the reaction mixture consisted of water, 50 mM MgCl2, Adeno_R mix (primer 300 nM, TaqMan probe 100 nM), Premix Ex Taq 2 and 15 ml of template, with a total volume of 50 ml. The PCR thermal prole consisted of an initial step at 95 C for 30 s followed by 35 cycles of 15 s at 95 C, 30 s at 58 C and 10 s at 72 C. Amplication 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 uorescence 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 specic PCR.

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G.L. Piacentini et al. 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). Inuenza A was detected in the adenoids of one patient and inuenza B in the tonsils of another. None of the other tested viruses was found. The percentages of positive results for the investigated bacteria for tonsillar and adenoid ndings 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. inuenzae were detected in 30 patients (54.5%); these bacteria were detected in 20 of the 33 adenoid samples (60.6%) and in 14 of the 30 tonsil samples (46.7%) (NS). Moraxella catarrhalis and H. inuenzae 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

Chromosomal DNA was extracted by a commercial kit (Roche Diagnostics, Germany). As regards PCR amplication, to conrm the extraction each DNA sample was tested for its ability to be amplied with B-globin specic primers [6]. For the detection of C. pneumoniae by nested PCR,primers that amplify the 207 bp fragment of the major outer membrane protein genes (ompA) were used [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. inuenzae, Moraxella catarrhalis and Streptococcus pneumoniae, the bacterial 16S rRNA gene was chosen as target and one common lower primer and three species-specic upper primers were used in a multiplex PCR [9]. Primers that amplify 504 bp fragment oprL gene were used for the detection of Pseudomonas aeruginosa [10]. Primers that amplify M protein gene were used for the detection of GABHS [11]. For Legionella pneumophila the amplied gene in nested PCR was macrophage infectivity potentiator protein (MIP) [12]. After amplication, 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.

70 60 50 40 30 20 10 0 GABHS M. catarrhalis H. influenzae C. pneumoniae Adenoids + tonsils (n=8) Tonsils (n=22) Adenoids (n=25) M. pneumoniae

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

Paediatric adenotonsils requiring adenotonsillectomy

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70 60 50 40 30 20 10 0 GABHS+MP GABHS+CP GABHS+others Adenoids + tonsils (n=8) Tonsils (n=22) Adenoids (n=25)

Figure 2. Details of distribution of contemporary agents.

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 GABHS and Moraxella catarrhalis or H. inuenzae and in 11 patients (20%) both GABHS and Mycoplasma pneumoniae or C. pneumoniae were detected. GABHS was signicantly more frequently found than viruses (p < 0.001) and more commonly than atypical bacteria when considered globally (p = 0.001). The details of distribution of the contemporary agents are reported in Figure 2. There was no signicant difference between the prevalence of viral genomic identication and that of Mycoplasma pneumoniae and C. pneumoniae. No signicant effect of the age and sex distribution of the patients was observed with respect to the detected agents.

Discussion PCR assay for the detection of genetic material from bacteria and viruses was used in the present study, because it has strong specicity, high sensitivity and

good correlation with culture ndings [6]. The detection of genetic material by PCR does not reect the viability of the organisms; however, it clearly increases the rate of identication of micro-organisms. The design of the present study, which was cross-sectional, did not allow us to follow up the changes in the titres of specic organisms, thus reducing the possibility of discriminating between actual infection and colonization or a previous infection in our study population. Nevertheless, serum assays might be used in acute conditions but are less useful to detect chronic infections with GABHS [7,8]. Furthermore, cultures are inuenced by previous antibiotic treatment, they may be difcult to perform and relatively insensitive in the case of Mycoplasma pneumoniae and C. pneumoniae. On the other hand PCR assay could detect also pathogens killed by previous antibiotic treatment. GABHS is the most important of the bacterial causes of acute pharyngitis [9] and the results of our study indicate that this is also the case for recurrent infections of adenoids and tonsils. In this study, Moraxella catarrhalis and H. inuenzae were more frequently found as single isolates (54.5%) than in association with GABHS (32.7%) (p = 0.034). This nding was in contrast with a recent observation showing an increased recovery of Moraxella catarrhalis and H. inuenzae in association with GABHS in children with acute pharyngotonsillitis [10]. Komaroff and co-workers [2] demonstrated significant serological evidence of infection by atypical bacteria, thus suggesting that potentially treatable

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G.L. Piacentini et al. of colonization by atypical bacteria in a general population 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 aetiological agents for the recurrent episodes of infection that suggested the opportunity for a tonsillectomy or adenoidectomy. Therefore, in consideration of the tendency for atypical bacteria to persist at the site of infection and to induce chronic inammation, we suggest consideration of their identication and appropriate treatment in children with recurrent upper airway illness. Further studies with a longitudinal design and a control group undergoing adenoidectomy 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 of antibiotic therapy. Declaration of interest: The authors report no conicts of interest. The authors alone are responsible for the content and writing of the paper. References
[1] Brook I. Pathogenicity and therapy of anaerobic bacteria in upper respiratory tract infections. Pediatr Infect Dis J 1987;6:131. [2] Komaroff AL, Aronson MD, Pass TM, Ervin CT, Branch WT Jr, Schachter J. Serologic evidence of chlamydial and mycoplasmal pharyngitis in adults. Science 1983; 222:9279. [3] Esposito S, Bosis S, Begliatti E, Droghetti R, Tremolati E, Tagliabue C, et al. Acute tonsillopharyngitis associated with atypical bacterial infection in children: natural history and impact of macrolide therapy. Clin Infect Dis 2006;43:2069. [4] Charnock DR, Chapman GD, Taylor RE, Wozniak A. Recurrent tonsillitis. The role of Chlamydia and Mycoplasma. Arch Otolaryngol Head Neck Surg 1992;118:5078. [5] Falck G, Heyman L, Gnarpe J, Gnarpe H. Chlamydia pneumoniae and chronic pharyngitis. Scand J Infect Dis 1995;27:17982. [6] Eisestein BI. New molecular techniques for microbial epidemiology and the diagnosis of infectious diseases. J Infect Dis 1990;161:595602. [7] Kaplan EL, Ferrieri P, Wannamaker L. Comparison of the antibody response to streptococcal cellular and extracellular antigens in acute pharyngitis. J Pediatr 1974;84:218. [8] Kaplan EL. The group A streptococcal upper respiratory tract carrier state: an enigma. J Pediatr 1980;97:33745. [9] Gerber MA. Diagnosis and treatment of pharyngitis in children. Pediatr Clin North Am 2005;52:72947. [10] Brook I, Gober AE. Increased recovery of Moraxella catarrhalis and Haemophilus inuenzae in association with group A beta-haemolytic streptococci in healthy children and those with pharyngotonsillitis. J Med Microbiol 2006;55:98992.

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 bacteria can 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 PCRpositive for Mycoplasma pneumoniae and 20% for C. pneumoniae. As a consequence, approximately 30% of our patients showed the presence of genetic material from atypical pathogens in adenoids and tonsils, which represents a clinically signicant proportion of patients and it gives support to the results of the previous study showing the presence of atypical bacteria 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 fourfold increase in specic 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]. The nding of similar prevalence in adults, studied during the acute phase of disease, and in children studied throughout the chronic phase of illness, is not surprising. In fact, C. pneumoniae infection is characterized 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 represented 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 recurrent URTI rather than having a pathogenetic effect. Unfortunately, the main limitations of the experimental design of this study were the lack of a control group of children undergoing adenoidectomy and tonsillectomy for airway obstruction in the absence of significant 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

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[11] Pruksakorn S, Sittisombut N, Phornphutkul C, Pruksachatkunakorn C, Good MF, Brandt E. Epidemiological analysis of non-M-typeable group A Streptococcus isolates from a Thai population in northern Thailand. J Clin Microbiol 2000;38:12504. [12] Bernander S, Hanson HS, Johansson B, von Stedingk LV. A nested polymerase chain reaction for detection of Legionella pneumophila in clinical specimens. Clin Microbiol Infect 1997;3:95101.

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[13] Zhang G, Ning B, Li Y. Detection of Chlamydia pneumoniae DNA in nasopharyngolaryngeal swab samples from patients with rhinitis and pharyngolaryngitis with polymerase chain reaction. Chin Med J 2000;113:1813. [14] Tjhie JHT, van Kuppeveld FJM, Roosendaal R, Melchers WJG, Gordijn R, MacLaren DM, et al. Direct PCR enables detection of Mycoplasma pneumoniae in patients with respiratory tract infections. J Clin Microbiol 1994;32:1116.

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