APMIS 123: 523–529
© 2015 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12383
Microbes of the tonsils in PFAPA (Periodic Fever, Aphtous stomatitis, Pharyngitis and Adenitis) syndrome – a possible trigger of febrile episodes ULLA LANTTO,1 PETRI KOIVUNEN,1 TERHI TAPIAINEN,2 VIRPI GLUMOFF,3 PASI HIRVIKOSKI,4 MATTI UHARI2 and MARJO RENKO2 Departments of 1Otorhinolaryngology, 2Pediatrics, Oulu University Hospital and Medical Research Center, University of Oulu, Oulu; 3Institute of Diagnostics, Department of Medical Microbiology and Immunology, University of Oulu, Oulu; and 4Department of Pathology, Oulu University Hospital, Oulu, Finland
Lantto U, Koivunen P, Tapiainen T, Glumoff V, Hirvikoski P, Uhari M, Renko M. Microbes of the tonsils in PFAPA (Periodic Fever, Aphtous stomatitis, Pharyngitis, and Adenitis) syndrome – a possible trigger of febrile episodes. APMIS 2015; 123: 523–529. Periodic Fever, Aphtous stomatitis, Pharyngitis, and Adenitis (PFAPA) is a childhood febrile syndrome that is often cured by tonsillectomy (TE). We hypothesized that microbes present in the tonsils may act as a trigger for the activation of inflammasomes and investigated the microbiology of the tonsils in PFAPA patients and controls. We recruited 31 consecutive children who underwent TE due to PFAPA; 24 children who underwent TE due to other reasons served as controls. We cultured all the samples for bacteria, mycobacteria, yeasts, and viruses and used PCR for 15 viruses. Also biofilm formation and histologic findings were identified. The samples of the patients yielded Candida albicans more often than did the controls (16 vs 0%, p = 0.003). Staphylococcus aureus occurred in only 10% of the patients, but in 38% of the controls (p = 0.01). Varicella zoster and Herpes simplex viruses occurred less often in patients than in controls. Biofilm was present in 55% of PFAPA tonsils, but in only 24% of the controls (p = 0.03). The microbes found in the tonsils of PFAPA patients showed significant differences from those of controls. This may in part explain the efficacy of TE in PFAPA. Key words: Recurrent fever; childhood; tonsillectomy; inflammasome; colonization. Marjo Renko, Department of Pediatrics, University of Oulu, Box 5000, Oulu FIN-90014, Finland. e-mail: marjo.renko @oulu.fi
Periodic Fever, Aphtous stomatitis, Pharyngitis, and Adenitis (PFAPA) is a childhood febrile syndrome in which the fever episodes occur in regular cycles of 3 to 5 weeks. Between febrile episodes, the patients are asymptomatic (1, 2). The symptoms usually resolve spontaneously before adolescence, but in recent years the syndrome has been described also among adults (3–5). The cumulative incidence of PFAPA has been calculated to be about two per 10 000 children up to 5 years of age (6), making PFAPA the most common periodic childhood febrile syndrome. The etiology and pathogenesis of PFAPA are unknown. Acute phase parameters such as leuko-
Received 15 January 2014. Accepted 12 February 2015
cyte counts, the erythrocyte sedimentation rate, Creactive protein, and the excretion of proinflammatory cytokines increase during the fever episodes (7–9). A single dose of corticosteroid rapidly reduces the fever, but this does not prevent subsequent episodes (2, 10). Recent studies support the hypothesis that PFAPA syndrome is a disease with dysregulated IL-1b production (11–13). The regulation of IL-1b production is closely related to a molecular complex known as inflammasome, which is activated by environmental stimuli, such as the presence of microbes (14, 15). In randomized controlled studies, tonsillectomy (TE) appears to be a curative treatment for PFAPA syndrome (16, 17), but the mechanism of this effect is unclear. We hypothesized that the microbiology of the tonsils in PFAPA patients might be 523
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different from that of healthy children which could then lead to triggering of the inflammasomes and the symptoms of PFAPA. We designed a prospective case control study in which we compared the microbiology, biofilm formation and histology of the tonsils removed from PFAPA patients and controls who underwent TE due to obstructive symptoms.
PATIENTS AND METHODS Between March 2006 and April 2010, we recruited 31 consecutive children who underwent TE due to PFAPA. The diagnostic criteria were the same as in our previous randomized controlled study of TE in PFAPA, i.e. at least five clockwisely reoccurring fever episode with possible signs or symptoms in the pharynx and cervical lymphnodes without any infectious or other obvious reason in an otherwise healthy child (16). During the same period, 24 children undergoing TE due to the clinical suspicion of obstructive sleep apnea and hypertrophied tonsils served as controls. We performed the TEs in the Department of Otorhinolaryngology of the Oulu University Hospital, Finland, using cold dissection with monopolar electrocauterization to obtain hemostasis. We then split the removed tonsils into halves in the operation room and sent them for further histologic and microbiologic examinations. For conventional microbiologic analyses the samples were processed immediately and for viral PCR studies after freezing. All analyses were carried out blinded to indications for TE. None of the patients had received cimetidine or corticosteroids pre- or postoperatively. The parents provided their written informed consent, and we collected data on the child’s symptoms before the surgery using a questionnaire. Previous use of any antimicrobials within 12 months before TE was obtained from the Finnish national Drug Purchase Register maintained by the Social Insurance Institution of Finland (KELA). The Ethics Committee of the Northern Ostrobothnia Hospital District, Oulu, Finland, found the study plan ethically acceptable.
nized in 800 lL of binding buffer containing poly-A RNA and proteinase K from the kit. The samples were then incubated for 30 min at 72 °C and centrifuged for 10 min at 80009 g. The supernatant was handled thereafter according to the manufacturer’s instructions. Parainfluenza-, influenza-, RS-, entero-, rhino-, and adenovirus were detected according to Kristo et al. (18). Metapneumovirus (MPV) was amplified with MPVN primers (19) and detected with a DIG-labeled probe 50 GTGTGTCTGGTGCTGAGG-DIG-30 . Herpes simplex (HSV) 1, HSV 2 and Varicella zoster virus (VZV) were detected according to Hukkanen and Vuorinen (20). Realtime-PCR for CMV, HHV6, and EBV were performed according to J€ a€ askel€ ainen et al. (21), and HPV according to Payan et al. (22). For analyses, we combined the results of all these microbial techniques and categorized the microbes as either present or absent.
Scanning electron microscopy Biofilms of the tonsil surface were visualized with a scanning electron microscope (SEM) and categorized as either present or absent (Fig. 1). Tissue samples for SEM were available from all PFAPA patients and 21 controls. A tissue sample with a diameter of 0.5–1 cm was taken for SEM. The sample was first rinsed with 0.9% NaCl to remove blood and nonadherent microbes. The sample was then ori-
A
Histologic examinations The tonsils were fixed in 10% buffered formalin and embedded in paraffin, and 5-lm thick sections were stained with hematoxyllin and eosin. In addition to the routine evaluation of histologic features, a special reference focused on active inflammation (microabscesses and crypt-infiltrating neutrophils). Furthermore, we recorded imprints consistent with the morphology of Actinomyceslike organisms.
Microbiologic examinations We used conventional methods to culture the samples for bacteria, mycobacteria, yeasts, and viruses. For PCR of the viruses, we used the High Pure Viral Nucleic Acid Isolation Kit (Roche Applied Science, Mannheim, Germany) to purify total nucleic acids from 100 lg of tonsillar tissue stored frozen at 70 °C. The frozen tissue was homoge-
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Fig. 1. Tonsil surface with (A) and without (B) biofilm formation in scanning electron microscopy. © 2015 APMIS. Published by John Wiley & Sons Ltd
MICROBES OF THE TONSILS IN PFAPA SYNDROME
ented by placing the specimen on a piece of cardboard. The specimen was then fixed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer. After fixation, the sample was briefly rinsed with phosphate-buffered saline and then with sterilized aqua. The specimen was dehydrated by placing it in 30% ethanol for 15 min, then in 50% ethanol for another 15 min, and finally in 70% ethanol. After the ethanol series, the specimen was processed with a Critical Point Dryer CPD 030 (Leica-Microsystems, Wetzlar, Germany) and given a platinum coating with a High Resolution Sputter Coater (Agar Scientific, Essex, UK). All samples were evaluated with a Zeiss Ultra plus FESEM (field emission scanning electron microscope, Cambridge, UK) at the Center of Microscopy and Nanotechnology at the University of Oulu. Trained evaluators who were blinded to indications for TE later evaluated all the samples.
Statistical methods
Table 1. Demographic characteristics and surgeries performed on 31 children with PFAPA syndrome and 24 controls PFAPA Controls (N = 31) (N = 24) 3.4 (1.7–18.2) 5.8 (2.7–15.1) Age at the time of surgery, median (range), years 2.4 (0.1–16.5) Age when symptoms begun, median (range), years Gender, boys, N (%) 18 (58%) 8 (33%) Tonsillectomy without 19 (61%) 4 (17%) adenoidectomy, N (%) Adenotonsillectomy, 11 (35%) 17 (71%) N (%) Adenoidectomy 1 (3%) 3 (13%) earlier, N (%) Antimicrobial courses mean (SD) Within 12 months 2.4 (2.5) 1.4 (1.9) before TE Within 3 months 0.6 (0.9) 0.3 (0.9) before TE1 Amoxicillin 0.17 (0.46) 0.21 (0.51) Penicillin 0.13 (0.43) 0.0 (0.0) Cephalexin 0.13 (0.43) 0.0 (0.0) Azithromycin 0.13 (0.57) 0.0 (0.0) 1 In addition one child received amoxicillin-clavulanate in control group, one child trimethoprim-sulfamethoxazole and clarithromycin in both PFAPA and control group, and one child clarithromycin in PFAPA group.
We used IBM SPSS Statistics 18.0 (Amonk, NY, USA) and STATA (College Stations, TX, USA) for statistical analyses and compared the findings between the PFAPA patients and controls. We calculated absolute differences and their 95% confidence intervals (CI) and used the standard normal deviate test (SND) to analyze the statistical significance of differences between the proportions. For continuous variables means (SD) or medians (range) were calculated in each group and the statistical significances of the differences were tested either with Student’s T-test or Mann–Whitney U according to the distributions. As the distribution of the number of antimicrobial courses before TE was skewed, the statistical significance of the difference between the groups was tested with Mann–Whitey U test. Because a child’s age can influence the microbiology of the tonsils and the sample size was too small to allow analyses on age groups, we performed forward stepwise logistic regression analyses, adjusting for age and gender, and calculated odds ratios (OR) and CIs for individual microbes.
number of antimicrobial courses 2.4) than in the control group (1.4) but the differences were not statistically significant (95% CI for the difference 0.25 to 2.2, p = 0.09) (Table 1).
RESULTS
Microbes of the PFAPA and control tonsils
Demographics of the cases and controls
The median age of the PFAPA patients at the onset of the fever periods was 2.4 years. The average duration of the PFAPA symptoms before TE was 12 months. The mean maximum fever was 39.7 °C, and the mean duration of the febrile episodes was 3.9 days. The mean time interval between two subsequent febrile episodes (from beginning to beginning) was 26 days. At the time of TE, the median age of the children in the PFAPA group was 3.4 years, and in the control group, 5.8 years (Table 1). In a follow-up of 6 months all the children with PFAPA had recovered from their symptoms after TE. Number of antimicrobial courses before TE
The exposure to antimicrobials within 12 months before TE was higher in PFAPA group (mean © 2015 APMIS. Published by John Wiley & Sons Ltd
Altogether 68% (21/31) of the PFAPA tonsils and 83% (20/24) of the control tonsils yielded at least one species of pathogenic bacteria (difference 16%, 95% CI 8 to 37%, p = 0.15). Haemophilus influenzae (H. influenzae) was the most common finding in both groups (Table 2). Streptococcus pneumoniae (S. pneumoniae) was more common in the PFAPA tonsils (23%) than in the controls (4%, Table 2). In contrast, the control samples more often tested positive for Staphylococcus aureus (S. aureus) than did the PFAPA cases (38 vs 10%, Table 2). At least one virus was found in either the viral culture or the PCR of 74% (23/31) of the PFAPA tonsils and 83% of the controls (20/24, difference 9%, 95% CI 14 to 30, p = 0.36) (Table 2). The control samples more often tested positive for VZV and HSV than did the PFAPA samples (Table 2). In the yeast culture, the tonsillar tissue of the PFAPA patients yielded C. albicans more often than did the controls (16 vs 0%, p = 0.003, Table 2); we
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Table 2. Microbiologic findings in the tonsils of 31 PFAPA patients and 24 controls PFAPA, N = 31 Controls, N = 24 Difference (CI) p Biofilm () 17 (55%) 5 (24%)2 31.0% (3.5 to 53.4%) 0.03 Biofilm1 Bacteria Haemophilus influenzae 12 (39%) 9 (38%) 1.2% (24.6 to 26.2%) 0.99 S. pneumoniae 7 (23%) 1 (4%) 18.4% (0.7 to 36.7%) 0.07 Fucobacteriae 6 (19%) 2 (8%) 11% (9.4 to 29.9%) 0.28 Prevotella 4 (13%) 1 (4%) 8.7% (9.2 to 25.7%) 0.22 S. aureus 3 (10%) 9 (38%) 28.8% (49.6 to 5.8%) 0.01 Streptococcus, group G 3 (10%) 4 (17%) 7.0% (28.0 to 11.6%) 0.45 Streptococcus, group C 2 (7%) 1 (4%) 2.2% (14.9 to 17.5%) 0.99 Streptococcus, group A 1 (3%) 2 (8%) 5% (23.3 to 9.3%) 0.32 Mycobacteriae 1 (3%) 0 3% (11.0 to 16.4 %) 0.999 Fungi C. albicans 5 (16%) 03 16.1% (1.1 to 32.8%) 0.003 Viruses4 Human herpesvirus 6 16 (53%)3 14 (58%) 5.0% (30.5 to 21.4%) 0.60 3 Adenoviruses 14 (47%) 7 (29%) 17.5% (9.0 to 41.2%) 0.18 Epstein Barr virus 8 (27%)3 9 (38%) 10.8% (35.4 to 14.0%) 0.40 Enteroviruses 4 (13%) 5 (21%) 7.9% (30.0 to 12.3%) 0.32 Varicella zoster virus 03 5 (21%) 20.8% (40.7 to 8.0%). 0.007 Herpes simplex 1 03 1 (4.2%) 4.2% (20.5 to 7.7%) 0.22 Herpes simplex 2 03 1 (4.2%) 4.2% (20.5 to 7.7%) 0.22 Influenza A virus 03 1 (4%) 4.2% (20.5 to 7.7%) 0.22 1 Biofilm on the surface of the tonsils was evaluated with scanning electron microscopy. 2 Three missing samples. 3 One missing sample. 4 No Cytomegalo-, Influenza B, Metapneumo-, Parainfluenza 1–3, Respiratory syncytial or rhinoviruses were found in PFAPA or control tonsil samples.
detected no other fungal organisms. In the mycobacterial culture, one tonsil sample in the PFAPA group tested positive for Mycobacterium bohemicum. Biofilm formation
In the scanning electron microscopy of the tonsil biopsies, we found biofilm on the surface of 55% of the PFAPA tonsils, but on only 24% of the controls (p = 0.03). S. pneumoniae proved more common in the biofilm-positive (6/22, 27%) than in the biofilm-negative tonsils (2/30, 7%, p = 0.04). The expression of other microbes showed no association with biofilm formation. S. pneumoniae and S. aureus seemed to emerge mostly in different patients, and only one control patient tested positive for both. This was also true for C. albicans and S. aureus (data not shown). Adjusted analyses
After adjusting the analyses for age and gender, the differences in S. aureus (OR 0.15, 95% CI 0.03 to 0.76), biofilm formation (OR 4.24, 95% CI 1.1 to 15.8), and VZV (OR 0.09, 95% CI 0–0.71) between the PFAPA group and the controls remained statistically significant. Samples from patients with respiratory symptoms
Four of the PFAPA patients and two of the controls exhibited symptoms of a common respiratory 526
infection (rhinitis or sore throat, but not fever) at the time of TE. One sample of these PFAPA tonsils obtained during respiratory infection yielded Streptococcus pyogenes, one S. pneumoniae and adenovirus, and one H. influenzae and enterovirus. The sample from one of the two controls with respiratory symptoms during TE contained H. influenzae, S. aureus, HSV2 and adenovirus, and the sample from the other control showed no pathogens. None of the tonsil samples were obtained during the acute febrile episode due to PFAPA. Histology
PFAPA cases and controls showed no differences in their routine histology findings. All the tonsils showed histologic features consistent with reactive follicular lymphatic tissue or follicular hyperplasia. We detected no foci of apparent necrosis or granulomatous reaction. We also noted active inflammation (neutrophils) in 10 (18%) samples with no predisposition to PFAPA (16% cases and 21% controls, difference 4%, 95% CI 16 to 24%, p = 0.65). Moreover, we detected particles consistent with the morphology of Actinomyces-like organisms in 15 (27%) samples: 5 PFAPA cases and 10 controls (16% vs 42%, difference 25%, 95% CI 2 to 48%, = 0.035). However, after adjusting this analysis for age and gender in logistic regression analysis, the difference was no more statistically significant. Additional immunohistochemical staining for B and T cells (CD20 and CD3) © 2015 APMIS. Published by John Wiley & Sons Ltd
MICROBES OF THE TONSILS IN PFAPA SYNDROME
showed expression patterns consistent with reactive lymphatic tissue in most cases (data not shown).
DISCUSSION This controlled prospective study showed marked differences in the microbiology of tonsils in PFAPA syndrome patients as compared to that of tonsils in controls. C. albicans, pneumococci and abundant biofilm formation were more common in tonsils removed from PFAPA patients than in those removed from controls; in contrast, samples from PFAPA patients yielded S. aureus and VZV less often. This finding is interesting in light of the present hypothesis that PFAPA is a disease with dysregulated function of an inflammasome, a molecular complex reacting, among other things, to the presence of microbes (12, 13). Microbes protect themselves from the host’s immune system and antimicrobials by forming complicated organized constructions called biofilms. Because antimicrobials are ineffective against biofilms, infections caused by biofilms usually require removal of the infection focus. Biofilm formations are abundant in hypertrophied tonsils and occur even more often in chronic tonsillitis (23–27). Removal of the tonsils has been suggested to reduce biofilm formation in the nasopharynx (28). However, we found even more often biofilm formation in the tonsils of PFAPA patients than in those of controls with tonsillar hypertrophy. Our approach to visualizing biofilms was morphologic, and we provided no further microbial description of the differences between the biofilms of PFAPA and control tonsils. Still, the effect of TE on PFAPA syndrome may depend on removal of the biofilms. Bacterial species colonizing the mucous membranes interact with each other by competing for resources and by producing chemicals or by inducing immune responses in the host (29). In our study, S. pneumoniae was a common finding, especially in biofilm-positive and S. aureus-negative PFAPA tonsils. A similar competition between viral and bacterial pathogens has been described in nasopharyngeal samples taken during and between acute otitis media episodes (30–32). Interaction between microbes, especially in polymicrobial biofilm societies, extends from prokaryotics to eukaryotics. Studies have shown that the presence of hyphenal forms of C. albicans may enhance the virulence and attachment ability of S. aureus, whereas in other bacteria, the effect can be the opposite (33, 34). In our series, C. albicans and S. aureus were present in different individuals.
© 2015 APMIS. Published by John Wiley & Sons Ltd
Inflammasomes are cytosolic protein complexes that are involved in activating the production of proinflammatory cytokines and caspase-1 after recognizing microbial and other environmental antigens. Specific microbes can lead to different pathogenmediated activation of inflammasomes. For example, C. albicans can trigger inflammasomes in filamentous, but not in unicellular yeast form (14), and pneumolysin expression is essential for S. pneumoniae to stimulate cytokine production (15). Rehaume et al. (35) have proposed a molecular link between Candida and auto-inflammatory diseases, suggesting that transformations in the activation of inflammasomes may either confer resistance to disseminating Candida infections or enhance mucosal colonization leading to inflammatory lesions. In our series, C. albicans was present in 5 of 31 PFAPA patients, but in none of the controls. The histology of the tonsils showed no specific changes in PFAPA patients. Most of the tonsils showed features of reactive follicular lymphatic hyperplasia, reflecting chronic tonsillitis regardless of the indication for TE. The histologic findings were quite similar, as described earlier about PFAPA tonsils (36). Additional immunohistochemical staining for B and T cells (CD20 and CD3) showed an expression pattern consistent with reactive lymphatic tissue reported earlier (36). Active inflammation showed no association with PFAPA, but TE was not performed during the hot/active phase of the disease. The strength of this study is its comprehensive microbiologic analysis of the tonsils. The high carriage rates of respiratory pathogens in this study are in line with those of previous findings on both bacteria (37) and viruses (38). In one previous case series of nine PFAPA patients who underwent TE, atypical bacteria and respiratory viruses were searched by molecular analyses from the removed tonsil tissue. Only one sample there showed positive result yielding adenovirus (39). One limitation of our study was that the controls were slightly older than the PFAPA cases. Because age influences both the colonization of the nasopharynx and the occurrence of PFAPA, we adjusted all the analyses with logistic regression modeling. However, most of the differences in the microbiologic findings were independent of age. In our study, PFAPA patients received more antibiotics than the controls did but the difference was smaller than expected. Previously S. aureus has been found both in the removed tonsils of recurrent tonsillitis (30%) and hypertrophied tonsils (23%) even though the patients with recurrent tonsillitis have probably had more courses of antimicrobials (40). Exposure to antimicrobials may affect the microbiologic findings, but it is not
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known how much and for how long time antibiotics change the flora of the tonsil tissue. In conclusion, the microbes on the tonsils of PFAPA patients in our controlled study differed markedly. In particular, C. albicans, pneumococci, and abundant biofilm formation on the surfaces of the removed tonsils were more common among the PFAPA patients than among the control patients. Because TE is an effective treatment for PFAPA syndrome, the evanescence of the periodic febrile episodes after TE may be due to the removal of the triggering microbial factor, in addition to immunomodulation.
CONFLICTS OF INTEREST
10.
11.
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The authors declare that they have no conflict of interest. 14. This work was supported by the P€ aivikki and Sakari Sohlberg Foundation, the Paulo Foundation, and the Foundation for Pediatric Research. The authors thank Birgitta Grekula for her technical assistance.
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23. Chole RA, Faddis BT. Anatomical evidence of microbial biofilms in tonsillar tissues: a possible mechanism to explain chronicity. Arch Otolaryngol Head Neck Surg 2003;129:634–6. 24. Kania RE, Lamers GE, Vonk MJ, Huy PT, Hiemstra PS, Bloemberg GV, et al. Demonstration of bacterial cells and glycocalyx in biofilms on human tonsils. Arch Otolaryngol Head Neck Surg 2007;133:115–21. 25. Al-Mazrou KA, Al-Khattaf AS. Adherent biofilms in adenotonsillar diseases in children. Arch Otolaryngol Head Neck Surg 2008;134:20–3. 26. Diaz RR, Picciafuoco S, Paraje MG, Villegas NA, Miranda JA, Albesa I, et al. Relevance of biofilms in pediatric tonsillar disease. Eur J Clin Microbiol Infect Dis 2011;30:1503–9. 27. Torretta S, Drago L, Marchisio P, Cappadona M, Rinaldi V, Nazzari E, et al. Recurrences in chronic tonsillitis substained by tonsillar biofilm-producing bacteria in children. Relationship with the grade of tonsillar hyperplasy. Int J Pediatr Otorhinolaryngol 2013;77:200–4. 28. Drago L, De VE, Torretta S, Mattina R, Marchisio P, Pignataro L. Biofilm formation by bacteria isolated from upper respiratory tract before and after adenotonsillectomy. APMIS 2012;120:410–16. 29. Margolis E, Yates A, Levin BR. The ecology of nasal colonization of Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus: the role of competition and interactions with host’s immune response. BMC Microbiol 2010;10:59. 30. Jacoby P, Watson K, Bowman J, Taylor A, Riley TV, Smith DW, et al. Modelling the co-occurrence of Streptococcus pneumoniae with other bacterial and viral pathogens in the upper respiratory tract. Vaccine 2007;25:2458–64. 31. Chien YW, Vidal JE, Grijalva CG, Bozio C, Edwards KM, Williams JV, et al. Density interactions among Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus in the nasopharynx of young Peruvian children. Pediatr Infect Dis J 2013;32:72–7.
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32. Xu Q, Almudervar A, Casey JR, Pichichero ME. Nasopharyngeal bacterial interactions in children. Emerg Infect Dis 2012;18:1738–45. 33. Shirtliff ME, Peters BM, Jabra-Rizk MA. Cross-kingdom interactions: Candida albicans and bacteria. FEMS Microbiol Lett 2009;299:1–8. 34. Peters BM, Jabra-Rizk MA, Scheper MA, Leid JG, Costerton JW, Shirtliff ME. Microbial interactions and differential protein expression in Staphylococcus aureus -Candida albicans dual-species biofilms. FEMS Immunol Med Microbiol 2010;59:493–503. 35. Rehaume LM, Jouault T, Chamaillard M. Lessons from the inflammasome: a molecular sentry linking Candida and Crohn’s disease. Trends Immunol 2010;31:171–5. 36. Peridis S, Koudoumnakis E, Theodoridis A, Stefanaki K, Helmis G, Houlakis M. Surgical outcomes and histology findings after tonsillectomy in children with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. Am J Otolaryngol 2010;31:472–5. 37. Garcia-Rodriguez JA, Fresnadillo Martinez MJ. Dynamics of nasopharyngeal colonization by potential respiratory pathogens. J Antimicrob Chemother 2002;50(Suppl S2):59–73. 38. Proenca-Modena JL, Pereira Valera FC, Jacob MG, Buzatto GP, Saturno TH, Lopes L, et al. High rates of detection of respiratory viruses in tonsillar tissues from children with chronic adenotonsillar disease. PLoS ONE 2012;7:e42136. 39. Pignataro L, Torretta S, Pietrogrande MC, Dellepiane RM, Pavesi P, Bossi A, et al. Outcome of Tonsillectomy in selected patients with PFAPA syndrome. Arch Otolaryngol Head Neck Surg 2009;135:548–53. 40. Jeong JH, Lee DW, Ryu RA, Lee YS, Lee SH, Kang JO, et al. Bacteriologic comparison of tonsil core in recurrent tonsillitis and tonsillar hypertrophy. Laryngoscope 2007;117:2146–51.
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Microbes of the tonsils in PFAPA (Periodic Fever, Aphtous stomatitis, Pharyngitis and Adenitis) syndrome – a possible trigger of febrile episodes ULLA LANTTO,1 PETRI KOIVUNEN,1 TERHI TAPIAINEN,2 VIRPI GLUMOFF,3 PASI HIRVIKOSKI,4 MATTI UHARI2 and MARJO RENKO2 Departments of 1Otorhinolaryngology, 2Pediatrics, Oulu University Hospital and Medical Research Center, University of Oulu, Oulu; 3Institute of Diagnostics, Department of Medical Microbiology and Immunology, University of Oulu, Oulu; and 4Department of Pathology, Oulu University Hospital, Oulu, Finland
Lantto U, Koivunen P, Tapiainen T, Glumoff V, Hirvikoski P, Uhari M, Renko M. Microbes of the tonsils in PFAPA (Periodic Fever, Aphtous stomatitis, Pharyngitis, and Adenitis) syndrome – a possible trigger of febrile episodes. APMIS 2015; 123: 523–529. Periodic Fever, Aphtous stomatitis, Pharyngitis, and Adenitis (PFAPA) is a childhood febrile syndrome that is often cured by tonsillectomy (TE). We hypothesized that microbes present in the tonsils may act as a trigger for the activation of inflammasomes and investigated the microbiology of the tonsils in PFAPA patients and controls. We recruited 31 consecutive children who underwent TE due to PFAPA; 24 children who underwent TE due to other reasons served as controls. We cultured all the samples for bacteria, mycobacteria, yeasts, and viruses and used PCR for 15 viruses. Also biofilm formation and histologic findings were identified. The samples of the patients yielded Candida albicans more often than did the controls (16 vs 0%, p = 0.003). Staphylococcus aureus occurred in only 10% of the patients, but in 38% of the controls (p = 0.01). Varicella zoster and Herpes simplex viruses occurred less often in patients than in controls. Biofilm was present in 55% of PFAPA tonsils, but in only 24% of the controls (p = 0.03). The microbes found in the tonsils of PFAPA patients showed significant differences from those of controls. This may in part explain the efficacy of TE in PFAPA. Key words: Recurrent fever; childhood; tonsillectomy; inflammasome; colonization. Marjo Renko, Department of Pediatrics, University of Oulu, Box 5000, Oulu FIN-90014, Finland. e-mail: marjo.renko @oulu.fi
Periodic Fever, Aphtous stomatitis, Pharyngitis, and Adenitis (PFAPA) is a childhood febrile syndrome in which the fever episodes occur in regular cycles of 3 to 5 weeks. Between febrile episodes, the patients are asymptomatic (1, 2). The symptoms usually resolve spontaneously before adolescence, but in recent years the syndrome has been described also among adults (3–5). The cumulative incidence of PFAPA has been calculated to be about two per 10 000 children up to 5 years of age (6), making PFAPA the most common periodic childhood febrile syndrome. The etiology and pathogenesis of PFAPA are unknown. Acute phase parameters such as leuko-
Received 15 January 2014. Accepted 12 February 2015
cyte counts, the erythrocyte sedimentation rate, Creactive protein, and the excretion of proinflammatory cytokines increase during the fever episodes (7–9). A single dose of corticosteroid rapidly reduces the fever, but this does not prevent subsequent episodes (2, 10). Recent studies support the hypothesis that PFAPA syndrome is a disease with dysregulated IL-1b production (11–13). The regulation of IL-1b production is closely related to a molecular complex known as inflammasome, which is activated by environmental stimuli, such as the presence of microbes (14, 15). In randomized controlled studies, tonsillectomy (TE) appears to be a curative treatment for PFAPA syndrome (16, 17), but the mechanism of this effect is unclear. We hypothesized that the microbiology of the tonsils in PFAPA patients might be 523
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different from that of healthy children which could then lead to triggering of the inflammasomes and the symptoms of PFAPA. We designed a prospective case control study in which we compared the microbiology, biofilm formation and histology of the tonsils removed from PFAPA patients and controls who underwent TE due to obstructive symptoms.
PATIENTS AND METHODS Between March 2006 and April 2010, we recruited 31 consecutive children who underwent TE due to PFAPA. The diagnostic criteria were the same as in our previous randomized controlled study of TE in PFAPA, i.e. at least five clockwisely reoccurring fever episode with possible signs or symptoms in the pharynx and cervical lymphnodes without any infectious or other obvious reason in an otherwise healthy child (16). During the same period, 24 children undergoing TE due to the clinical suspicion of obstructive sleep apnea and hypertrophied tonsils served as controls. We performed the TEs in the Department of Otorhinolaryngology of the Oulu University Hospital, Finland, using cold dissection with monopolar electrocauterization to obtain hemostasis. We then split the removed tonsils into halves in the operation room and sent them for further histologic and microbiologic examinations. For conventional microbiologic analyses the samples were processed immediately and for viral PCR studies after freezing. All analyses were carried out blinded to indications for TE. None of the patients had received cimetidine or corticosteroids pre- or postoperatively. The parents provided their written informed consent, and we collected data on the child’s symptoms before the surgery using a questionnaire. Previous use of any antimicrobials within 12 months before TE was obtained from the Finnish national Drug Purchase Register maintained by the Social Insurance Institution of Finland (KELA). The Ethics Committee of the Northern Ostrobothnia Hospital District, Oulu, Finland, found the study plan ethically acceptable.
nized in 800 lL of binding buffer containing poly-A RNA and proteinase K from the kit. The samples were then incubated for 30 min at 72 °C and centrifuged for 10 min at 80009 g. The supernatant was handled thereafter according to the manufacturer’s instructions. Parainfluenza-, influenza-, RS-, entero-, rhino-, and adenovirus were detected according to Kristo et al. (18). Metapneumovirus (MPV) was amplified with MPVN primers (19) and detected with a DIG-labeled probe 50 GTGTGTCTGGTGCTGAGG-DIG-30 . Herpes simplex (HSV) 1, HSV 2 and Varicella zoster virus (VZV) were detected according to Hukkanen and Vuorinen (20). Realtime-PCR for CMV, HHV6, and EBV were performed according to J€ a€ askel€ ainen et al. (21), and HPV according to Payan et al. (22). For analyses, we combined the results of all these microbial techniques and categorized the microbes as either present or absent.
Scanning electron microscopy Biofilms of the tonsil surface were visualized with a scanning electron microscope (SEM) and categorized as either present or absent (Fig. 1). Tissue samples for SEM were available from all PFAPA patients and 21 controls. A tissue sample with a diameter of 0.5–1 cm was taken for SEM. The sample was first rinsed with 0.9% NaCl to remove blood and nonadherent microbes. The sample was then ori-
A
Histologic examinations The tonsils were fixed in 10% buffered formalin and embedded in paraffin, and 5-lm thick sections were stained with hematoxyllin and eosin. In addition to the routine evaluation of histologic features, a special reference focused on active inflammation (microabscesses and crypt-infiltrating neutrophils). Furthermore, we recorded imprints consistent with the morphology of Actinomyceslike organisms.
Microbiologic examinations We used conventional methods to culture the samples for bacteria, mycobacteria, yeasts, and viruses. For PCR of the viruses, we used the High Pure Viral Nucleic Acid Isolation Kit (Roche Applied Science, Mannheim, Germany) to purify total nucleic acids from 100 lg of tonsillar tissue stored frozen at 70 °C. The frozen tissue was homoge-
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B
Fig. 1. Tonsil surface with (A) and without (B) biofilm formation in scanning electron microscopy. © 2015 APMIS. Published by John Wiley & Sons Ltd
MICROBES OF THE TONSILS IN PFAPA SYNDROME
ented by placing the specimen on a piece of cardboard. The specimen was then fixed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer. After fixation, the sample was briefly rinsed with phosphate-buffered saline and then with sterilized aqua. The specimen was dehydrated by placing it in 30% ethanol for 15 min, then in 50% ethanol for another 15 min, and finally in 70% ethanol. After the ethanol series, the specimen was processed with a Critical Point Dryer CPD 030 (Leica-Microsystems, Wetzlar, Germany) and given a platinum coating with a High Resolution Sputter Coater (Agar Scientific, Essex, UK). All samples were evaluated with a Zeiss Ultra plus FESEM (field emission scanning electron microscope, Cambridge, UK) at the Center of Microscopy and Nanotechnology at the University of Oulu. Trained evaluators who were blinded to indications for TE later evaluated all the samples.
Statistical methods
Table 1. Demographic characteristics and surgeries performed on 31 children with PFAPA syndrome and 24 controls PFAPA Controls (N = 31) (N = 24) 3.4 (1.7–18.2) 5.8 (2.7–15.1) Age at the time of surgery, median (range), years 2.4 (0.1–16.5) Age when symptoms begun, median (range), years Gender, boys, N (%) 18 (58%) 8 (33%) Tonsillectomy without 19 (61%) 4 (17%) adenoidectomy, N (%) Adenotonsillectomy, 11 (35%) 17 (71%) N (%) Adenoidectomy 1 (3%) 3 (13%) earlier, N (%) Antimicrobial courses mean (SD) Within 12 months 2.4 (2.5) 1.4 (1.9) before TE Within 3 months 0.6 (0.9) 0.3 (0.9) before TE1 Amoxicillin 0.17 (0.46) 0.21 (0.51) Penicillin 0.13 (0.43) 0.0 (0.0) Cephalexin 0.13 (0.43) 0.0 (0.0) Azithromycin 0.13 (0.57) 0.0 (0.0) 1 In addition one child received amoxicillin-clavulanate in control group, one child trimethoprim-sulfamethoxazole and clarithromycin in both PFAPA and control group, and one child clarithromycin in PFAPA group.
We used IBM SPSS Statistics 18.0 (Amonk, NY, USA) and STATA (College Stations, TX, USA) for statistical analyses and compared the findings between the PFAPA patients and controls. We calculated absolute differences and their 95% confidence intervals (CI) and used the standard normal deviate test (SND) to analyze the statistical significance of differences between the proportions. For continuous variables means (SD) or medians (range) were calculated in each group and the statistical significances of the differences were tested either with Student’s T-test or Mann–Whitney U according to the distributions. As the distribution of the number of antimicrobial courses before TE was skewed, the statistical significance of the difference between the groups was tested with Mann–Whitey U test. Because a child’s age can influence the microbiology of the tonsils and the sample size was too small to allow analyses on age groups, we performed forward stepwise logistic regression analyses, adjusting for age and gender, and calculated odds ratios (OR) and CIs for individual microbes.
number of antimicrobial courses 2.4) than in the control group (1.4) but the differences were not statistically significant (95% CI for the difference 0.25 to 2.2, p = 0.09) (Table 1).
RESULTS
Microbes of the PFAPA and control tonsils
Demographics of the cases and controls
The median age of the PFAPA patients at the onset of the fever periods was 2.4 years. The average duration of the PFAPA symptoms before TE was 12 months. The mean maximum fever was 39.7 °C, and the mean duration of the febrile episodes was 3.9 days. The mean time interval between two subsequent febrile episodes (from beginning to beginning) was 26 days. At the time of TE, the median age of the children in the PFAPA group was 3.4 years, and in the control group, 5.8 years (Table 1). In a follow-up of 6 months all the children with PFAPA had recovered from their symptoms after TE. Number of antimicrobial courses before TE
The exposure to antimicrobials within 12 months before TE was higher in PFAPA group (mean © 2015 APMIS. Published by John Wiley & Sons Ltd
Altogether 68% (21/31) of the PFAPA tonsils and 83% (20/24) of the control tonsils yielded at least one species of pathogenic bacteria (difference 16%, 95% CI 8 to 37%, p = 0.15). Haemophilus influenzae (H. influenzae) was the most common finding in both groups (Table 2). Streptococcus pneumoniae (S. pneumoniae) was more common in the PFAPA tonsils (23%) than in the controls (4%, Table 2). In contrast, the control samples more often tested positive for Staphylococcus aureus (S. aureus) than did the PFAPA cases (38 vs 10%, Table 2). At least one virus was found in either the viral culture or the PCR of 74% (23/31) of the PFAPA tonsils and 83% of the controls (20/24, difference 9%, 95% CI 14 to 30, p = 0.36) (Table 2). The control samples more often tested positive for VZV and HSV than did the PFAPA samples (Table 2). In the yeast culture, the tonsillar tissue of the PFAPA patients yielded C. albicans more often than did the controls (16 vs 0%, p = 0.003, Table 2); we
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Table 2. Microbiologic findings in the tonsils of 31 PFAPA patients and 24 controls PFAPA, N = 31 Controls, N = 24 Difference (CI) p Biofilm () 17 (55%) 5 (24%)2 31.0% (3.5 to 53.4%) 0.03 Biofilm1 Bacteria Haemophilus influenzae 12 (39%) 9 (38%) 1.2% (24.6 to 26.2%) 0.99 S. pneumoniae 7 (23%) 1 (4%) 18.4% (0.7 to 36.7%) 0.07 Fucobacteriae 6 (19%) 2 (8%) 11% (9.4 to 29.9%) 0.28 Prevotella 4 (13%) 1 (4%) 8.7% (9.2 to 25.7%) 0.22 S. aureus 3 (10%) 9 (38%) 28.8% (49.6 to 5.8%) 0.01 Streptococcus, group G 3 (10%) 4 (17%) 7.0% (28.0 to 11.6%) 0.45 Streptococcus, group C 2 (7%) 1 (4%) 2.2% (14.9 to 17.5%) 0.99 Streptococcus, group A 1 (3%) 2 (8%) 5% (23.3 to 9.3%) 0.32 Mycobacteriae 1 (3%) 0 3% (11.0 to 16.4 %) 0.999 Fungi C. albicans 5 (16%) 03 16.1% (1.1 to 32.8%) 0.003 Viruses4 Human herpesvirus 6 16 (53%)3 14 (58%) 5.0% (30.5 to 21.4%) 0.60 3 Adenoviruses 14 (47%) 7 (29%) 17.5% (9.0 to 41.2%) 0.18 Epstein Barr virus 8 (27%)3 9 (38%) 10.8% (35.4 to 14.0%) 0.40 Enteroviruses 4 (13%) 5 (21%) 7.9% (30.0 to 12.3%) 0.32 Varicella zoster virus 03 5 (21%) 20.8% (40.7 to 8.0%). 0.007 Herpes simplex 1 03 1 (4.2%) 4.2% (20.5 to 7.7%) 0.22 Herpes simplex 2 03 1 (4.2%) 4.2% (20.5 to 7.7%) 0.22 Influenza A virus 03 1 (4%) 4.2% (20.5 to 7.7%) 0.22 1 Biofilm on the surface of the tonsils was evaluated with scanning electron microscopy. 2 Three missing samples. 3 One missing sample. 4 No Cytomegalo-, Influenza B, Metapneumo-, Parainfluenza 1–3, Respiratory syncytial or rhinoviruses were found in PFAPA or control tonsil samples.
detected no other fungal organisms. In the mycobacterial culture, one tonsil sample in the PFAPA group tested positive for Mycobacterium bohemicum. Biofilm formation
In the scanning electron microscopy of the tonsil biopsies, we found biofilm on the surface of 55% of the PFAPA tonsils, but on only 24% of the controls (p = 0.03). S. pneumoniae proved more common in the biofilm-positive (6/22, 27%) than in the biofilm-negative tonsils (2/30, 7%, p = 0.04). The expression of other microbes showed no association with biofilm formation. S. pneumoniae and S. aureus seemed to emerge mostly in different patients, and only one control patient tested positive for both. This was also true for C. albicans and S. aureus (data not shown). Adjusted analyses
After adjusting the analyses for age and gender, the differences in S. aureus (OR 0.15, 95% CI 0.03 to 0.76), biofilm formation (OR 4.24, 95% CI 1.1 to 15.8), and VZV (OR 0.09, 95% CI 0–0.71) between the PFAPA group and the controls remained statistically significant. Samples from patients with respiratory symptoms
Four of the PFAPA patients and two of the controls exhibited symptoms of a common respiratory 526
infection (rhinitis or sore throat, but not fever) at the time of TE. One sample of these PFAPA tonsils obtained during respiratory infection yielded Streptococcus pyogenes, one S. pneumoniae and adenovirus, and one H. influenzae and enterovirus. The sample from one of the two controls with respiratory symptoms during TE contained H. influenzae, S. aureus, HSV2 and adenovirus, and the sample from the other control showed no pathogens. None of the tonsil samples were obtained during the acute febrile episode due to PFAPA. Histology
PFAPA cases and controls showed no differences in their routine histology findings. All the tonsils showed histologic features consistent with reactive follicular lymphatic tissue or follicular hyperplasia. We detected no foci of apparent necrosis or granulomatous reaction. We also noted active inflammation (neutrophils) in 10 (18%) samples with no predisposition to PFAPA (16% cases and 21% controls, difference 4%, 95% CI 16 to 24%, p = 0.65). Moreover, we detected particles consistent with the morphology of Actinomyces-like organisms in 15 (27%) samples: 5 PFAPA cases and 10 controls (16% vs 42%, difference 25%, 95% CI 2 to 48%, = 0.035). However, after adjusting this analysis for age and gender in logistic regression analysis, the difference was no more statistically significant. Additional immunohistochemical staining for B and T cells (CD20 and CD3) © 2015 APMIS. Published by John Wiley & Sons Ltd
MICROBES OF THE TONSILS IN PFAPA SYNDROME
showed expression patterns consistent with reactive lymphatic tissue in most cases (data not shown).
DISCUSSION This controlled prospective study showed marked differences in the microbiology of tonsils in PFAPA syndrome patients as compared to that of tonsils in controls. C. albicans, pneumococci and abundant biofilm formation were more common in tonsils removed from PFAPA patients than in those removed from controls; in contrast, samples from PFAPA patients yielded S. aureus and VZV less often. This finding is interesting in light of the present hypothesis that PFAPA is a disease with dysregulated function of an inflammasome, a molecular complex reacting, among other things, to the presence of microbes (12, 13). Microbes protect themselves from the host’s immune system and antimicrobials by forming complicated organized constructions called biofilms. Because antimicrobials are ineffective against biofilms, infections caused by biofilms usually require removal of the infection focus. Biofilm formations are abundant in hypertrophied tonsils and occur even more often in chronic tonsillitis (23–27). Removal of the tonsils has been suggested to reduce biofilm formation in the nasopharynx (28). However, we found even more often biofilm formation in the tonsils of PFAPA patients than in those of controls with tonsillar hypertrophy. Our approach to visualizing biofilms was morphologic, and we provided no further microbial description of the differences between the biofilms of PFAPA and control tonsils. Still, the effect of TE on PFAPA syndrome may depend on removal of the biofilms. Bacterial species colonizing the mucous membranes interact with each other by competing for resources and by producing chemicals or by inducing immune responses in the host (29). In our study, S. pneumoniae was a common finding, especially in biofilm-positive and S. aureus-negative PFAPA tonsils. A similar competition between viral and bacterial pathogens has been described in nasopharyngeal samples taken during and between acute otitis media episodes (30–32). Interaction between microbes, especially in polymicrobial biofilm societies, extends from prokaryotics to eukaryotics. Studies have shown that the presence of hyphenal forms of C. albicans may enhance the virulence and attachment ability of S. aureus, whereas in other bacteria, the effect can be the opposite (33, 34). In our series, C. albicans and S. aureus were present in different individuals.
© 2015 APMIS. Published by John Wiley & Sons Ltd
Inflammasomes are cytosolic protein complexes that are involved in activating the production of proinflammatory cytokines and caspase-1 after recognizing microbial and other environmental antigens. Specific microbes can lead to different pathogenmediated activation of inflammasomes. For example, C. albicans can trigger inflammasomes in filamentous, but not in unicellular yeast form (14), and pneumolysin expression is essential for S. pneumoniae to stimulate cytokine production (15). Rehaume et al. (35) have proposed a molecular link between Candida and auto-inflammatory diseases, suggesting that transformations in the activation of inflammasomes may either confer resistance to disseminating Candida infections or enhance mucosal colonization leading to inflammatory lesions. In our series, C. albicans was present in 5 of 31 PFAPA patients, but in none of the controls. The histology of the tonsils showed no specific changes in PFAPA patients. Most of the tonsils showed features of reactive follicular lymphatic hyperplasia, reflecting chronic tonsillitis regardless of the indication for TE. The histologic findings were quite similar, as described earlier about PFAPA tonsils (36). Additional immunohistochemical staining for B and T cells (CD20 and CD3) showed an expression pattern consistent with reactive lymphatic tissue reported earlier (36). Active inflammation showed no association with PFAPA, but TE was not performed during the hot/active phase of the disease. The strength of this study is its comprehensive microbiologic analysis of the tonsils. The high carriage rates of respiratory pathogens in this study are in line with those of previous findings on both bacteria (37) and viruses (38). In one previous case series of nine PFAPA patients who underwent TE, atypical bacteria and respiratory viruses were searched by molecular analyses from the removed tonsil tissue. Only one sample there showed positive result yielding adenovirus (39). One limitation of our study was that the controls were slightly older than the PFAPA cases. Because age influences both the colonization of the nasopharynx and the occurrence of PFAPA, we adjusted all the analyses with logistic regression modeling. However, most of the differences in the microbiologic findings were independent of age. In our study, PFAPA patients received more antibiotics than the controls did but the difference was smaller than expected. Previously S. aureus has been found both in the removed tonsils of recurrent tonsillitis (30%) and hypertrophied tonsils (23%) even though the patients with recurrent tonsillitis have probably had more courses of antimicrobials (40). Exposure to antimicrobials may affect the microbiologic findings, but it is not
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known how much and for how long time antibiotics change the flora of the tonsil tissue. In conclusion, the microbes on the tonsils of PFAPA patients in our controlled study differed markedly. In particular, C. albicans, pneumococci, and abundant biofilm formation on the surfaces of the removed tonsils were more common among the PFAPA patients than among the control patients. Because TE is an effective treatment for PFAPA syndrome, the evanescence of the periodic febrile episodes after TE may be due to the removal of the triggering microbial factor, in addition to immunomodulation.
CONFLICTS OF INTEREST
10.
11.
12.
13.
The authors declare that they have no conflict of interest. 14. This work was supported by the P€ aivikki and Sakari Sohlberg Foundation, the Paulo Foundation, and the Foundation for Pediatric Research. The authors thank Birgitta Grekula for her technical assistance.
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