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Aetiology of acute pharyngitis: the role of atypical bacteria

^1,2Institute of Paediatrics¹ and Institute of Respiratory Diseases, IRCCS Maggiore Hospital², University of Milan, Milan, Italy

Correspondence Nicola Principi Nicola.Principi{at}unimi.it

Received September 30, 2003
Accepted January 25, 2004

In order to establish the role of atypical bacteria and compare characteristics of different infectious agents in acute pharyngitis, 127 patients with acute pharyngitis (66 males; median age, 5.33 years; range, 6 months to 14 years) and 130 healthy subjects of similar sex and age were studied. Serology with paired samples and PCR on nasopharyngeal aspirates and throat cultures were used to identify bacteria and viruses. Viruses were identified in 43 patients (33.8 %) and five controls (3.8 %; P < 0.0001), potential bacterial pathogens in 34 patients (26.8 %) and 26 controls (20 %; P = 0.256) and mixed viral/bacterial pathogens in 26 patients (20.5 %) and none of the controls (P < 0.0001). The main aetiological agents were adenovirus, respiratory syncytial virus (RSV), Mycoplasma pneumoniae, Streptococcus pyogenes and Chlamydia pneumoniae. M. pneumoniae was the agent found most frequently as a single pathogen. A history of recurrent pharyngitis, having older siblings and a negative outcome were significantly more common among patients with acute M. pneumoniae infection than among those with infections due to other pathogens or healthy controls. This study demonstrates that: (i) adenovirus and RSV have a prominent role in acute pharyngitis; (ii) S. pyogenes is found frequently, but it is not possible to distinguish simple carriers from patients with a true infection; (iii) M. pneumoniae appears to be able to cause acute pharyngitis per se; and (iv) C. pneumoniae seems to be mainly a co-pathogen. To avoid the risk of an incorrect therapeutic approach, simple laboratory investigations that allow rapid identification of M. pneumoniae infections are urgently needed.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Acute pharyngitis is one of the most common childhood illnesses to be diagnosed in an outpatient setting (Bisno et al., 1997, 2002; Bisno, 2001). Viruses and Streptococcus pyogenes are considered to be the most frequent causes of this disease, but particular attention is usually given only to streptococcal cases, as they may be followed by severe early and late complications and are the only ones for which antibiotic treatment is definitely indicated (Bisno et al., 1997, 2002; Bisno, 2001).

It has recently been demonstrated that a significant part of non-streptococcal acute pharyngitis may be associated with Mycoplasma pneumoniae and/or Chlamydia pneumoniae infection, but it has not been established whether these atypical bacteria act simply as co-pathogens or as primary aetiological agents, nor is it known what the outcome of such infections is when they are not treated with antibiotics (Principi & Esposito, 2001; Esposito et al., 2002b). Definition of the real role of M. pneumoniae and C. pneumoniae in causing acute pharyngitis would be useful for deciding the best diagnostic and therapeutic approach. If these bacteria are primary causes of pharyngitis, and if the disease is likely to have a more complicated course unless treated with adequate antimicrobial agents, some cases that are not usually treated (as they are considered to be of viral origin) need to be identified and treated correctly. These bacteria can be eradicated safely in children only with macrolides (File et al., 1998; Principi & Esposito, 1999; Hammerschlag, 2000, 2001), which, at least in some geographical areas, are not always active against streptococcal cases (Cornaglia et al., 1998; Facinelli et al., 2001; Martin et al., 2002). On the other hand, penicillin V, amoxycillin and oral cephalosporins, which are currently considered to be the drugs of choice for streptococcal pharyngitis (Bisno et al., 2002), are not active against atypical bacteria (File et al., 1998; Principi & Esposito, 1999; Hammerschlag, 2000, 2001).

Although microbiological diagnostic methods have improved greatly over the last 20 years (Mäkelä et al., 1998; Heikkinen et al., 2002; Monto, 2002), no recent prospective and comprehensive study of the aetiology of acute pharyngitis has been published. The aim of this study was to evaluate the aetiology of acute pharyngitis in childhood in order to establish the role of atypical bacteria and compare the characteristics of infections due to different pathogens.

	METHODS

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Study population and follow-up.

The study was conducted at the primary care centre of the Institute of Paediatrics, University of Milan, Italy, between February 2000 and March 2002. The level of pharyngitis over time was not concurrent with epidemic peaks of viral or bacterial infections. During this period, we enrolled 127 patients aged between 6 months and 14 years (66 males; median age of all enrolled children, 5.33 years) with an acute episode of pure pharyngitis, defined as evidence of uvular and pharyngeal or tonsillar inflammation in the presence of fever, sore throat or dysphagia, without any signs or symptoms of lower respiratory tract infection (Kaplan & Johnson, 2001). Exclusion criteria included severe concomitant diseases (e.g. neoplasia, kidney or liver disease, immunodepression, cardiovascular disease or malabsorption syndrome) and systemic antibiotic treatment in the 48 h preceding study entry or therapy with azithromycin during the previous week or with intramuscular benzathine penicillin G during the previous month. During the same period, 130 healthy subjects of similar sex and age (67 males; median age, 5.11 years) without any history of respiratory tract infection or antibiotic treatment in the 3 months before enrolment, who were seen for minor surgical problems, were enrolled as a control group. The study protocol was approved by the institutional review board of the University of Milan and written informed consent was obtained from the parents or legal guardians of all participants.

Upon enrolment, systematic recordings were made of the children's demographic characteristics and medical history by using standardized written questionnaires (Principi et al., 1999; Esposito et al., 2002b). Questions included duration of breast-feeding, living conditions, childcare attendance, number and age of family members, birth rank, smoking habits of family members living together, number and type of respiratory infections in the previous 6 months, presence of a history of recurrent pharyngitis (defined as at least three acute episodes in the 6 months preceding enrolment) (Korppi, 1997; Esposito et al., 2002b) and number and type of antimicrobials administered during the previous 6 months.

After a complete physical examination, laboratory samples were taken from all patients and controls, including venous blood specimens for haematological and blood chemistry tests (haemoglobin, leukocyte differential count, erythrocyte sedimentation rate and C-reactive protein), acute serum for assay of antibodies to M. pneumoniae, C. pneumoniae, adenovirus, influenza A and B viruses, parainfluenza virus types 1, 2 and 3, respiratory syncytial virus (RSV), Epstein–Barr virus (EBV) and herpes simplex virus type 1 (HSV-1), nasopharyngeal aspirates for detection of M. pneumoniae and C. pneumoniae DNA and a pharyngeal swab for S. pyogenes culture. Children with acute pharyngitis whose throat swabs were positive for S. pyogenes received 50 mg amoxycillin kg^–1 day^–1 (divided between three daily doses) for 10 days, whereas other patients received only symptomatic therapy; no other antibiotic treatment was given during the study period to any patients without streptococcal infection and no therapy was administered to controls, even when throat swab results were positive for S. pyogenes. The children's caregivers were asked to complete a diary card concerning the child's signs and symptoms of pharyngitis and any other medical problems. Parents were also asked to return immediately to the study centre if the children experienced any persistent, recurrent or worsening signs or symptoms.

All children were scheduled to return on days 11–15 after enrolment for an additional visit, at which their signs and symptoms were assessed. Between 4 and 5 weeks after enrolment, medical history, general physical condition and clinical symptoms of each child were re-evaluated and a second serum sample was obtained, to assay convalescent antibody titres against M. pneumoniae, C. pneumoniae and viruses.

Each child's clinical outcome was defined at each visit as ‘positive’ (absence of signs and symptoms of pharyngitis) or ‘negative’ (persistence or progression of signs and symptoms of pharyngitis or development of new clinical findings consistent with acute pharyngitis) (Kaplan & Johnson, 2001; Principi et al., 2001; Esposito et al., 2002b).

Antibody assays and PCR for atypical bacteria.

Each acute and convalescent serum sample was tested after absorption for IgM and IgG antibodies to M. pneumoniae (ELISA; Pantec), and IgM, IgA and IgG antibodies to C. pneumoniae (microimmunofluorescence; Labsystems) as described previously (Wang & Grayston, 1970; Principi et al., 2001; Esposito et al., 2002a). Nasopharyngeal aspirates were evaluated for the presence of M. pneumoniae and C. pneumoniae DNA by using validated nested PCR for both pathogens, as described previously (Tong & Sillis, 1993; Abele-Horn et al., 1998; Blasi et al., 1999; Principi et al., 2001; Esposito et al., 2002a).

The nested PCR technique for M. pneumoniae was developed by Abele-Horn et al. (1998), and that for C. pneumoniae by Tong & Sillis (1993); both techniques have been found to correlate well with cultures (Tong & Sillis, 1993; Abele-Horn et al., 1998). To avoid risk of contamination, sample preparation, PCR amplification and product analysis were performed in separate rooms. Positive and negative controls were included in each assay. Negative controls contained all PCR reagents and sterile distilled water; in the case of M. pneumoniae, we also used DNA from the reference strains Mycoplasma orale T519, Mycoplasma salivarium A889 and Mycoplasma genitalium G37c, as described previously (Abele-Horn et al., 1998). Serial dilutions of purified M. pneumoniae and C. pneumoniae were used as positive controls for all runs in order to ensure successful nucleic acid amplification. All PCR-negative samples were analysed by PCR for the presence of ß-actin DNA, to confirm the presence of DNA in the samples.

Primers MP-1 and MP-2 were used for M. pneumoniae-specific amplification (Abele-Horn et al., 1998). For the first and second rounds of amplification, reactions were carried out in 50 µl volumes, with 0.1 µM each primer (Abele-Horn et al., 1998). Amplification was carried out for 40 cycles, each consisting of 20 s at 95 °C, 2 min at 63 °C and 1 min at 72 °C (Principi et al., 2001). For M. pneumoniae nested PCR, primers MUH-1 and MUH-2 were used (Abele-Horn et al., 1998). Nested amplification was performed by using 5 µl of a 1 : 10 dilution of PCR product (5 µl in 45 µl sterile water) from the first round of amplification, under identical conditions (Abele-Horn et al., 1998).

Nested touchdown PCR for detection of C. pneumoniae DNA was performed by using primers that were designed to detect the major outer-membrane protein (Tong & Sillis, 1993; Blasi et al., 1999). Extracted DNA solution (10 µl in a total volume of 50 µl) was used in the first round of PCR, then 5 µl PCR product that was amplified by the outer primers was transferred to a new PCR mix (total volume, 50 µl) for a second amplification using the inner primers (Blasi et al., 1999). The first round consisted of 40 cycles and the second of 35 cycles (Blasi et al., 1999).

Acute M. pneumoniae and/or C. pneumoniae infection was diagnosed if the child had a significant antibody response to one of the pathogens in paired sera (M. pneumoniae: an IgM-specific antibody titre of 1 : 100 or a fourfold increase in IgG antibody; C. pneumoniae: an IgM-specific antibody titre of 1 : 16 or a fourfold increase in IgG antibody) and/or if nasopharyngeal aspirates were PCR-positive, according to previously established criteria (Principi et al., 2001; Esposito et al., 2002a, b).

Viral antibody assays.

Acute and convalescent serum samples were also tested by ELISA for IgM and IgG antibodies to adenovirus, influenza A and B viruses, parainfluenza virus types 1, 2 and 3, RSV, EBV and HSV-1, according to the manufacturer's instructions (Pantec).

Acute adenovirus, influenza A and B viruses, parainfluenza viruses types 1, 2 and 3, RSV, EBV and/or HSV-1 infection was diagnosed if the child had a significant antibody response to one of the viruses in paired sera (for each virus, an IgM-specific antibody titre of 1 : 100 or a fourfold increase in IgG antibody), according to previously established criteria (Heiskanen-Kosma et al., 1998). For EBV, IgM and IgG antibodies against viral capsid antigen were evaluated.

S. pyogenes culture.

Throat cultures were obtained by swabbing both tonsils and the posterior pharynx with a rayon-tipped swab (BBL Becton Dickinson). The swab was then placed in Amies’ medium without charcoal and plated within 2 h onto 5 % sheep's blood agar; the agar was stabbed and a bacitracin disc was placed in the primary streak (BBL Becton Dickinson). Plates were incubated at 37 °C in 5 % carbon dioxide and were examined after 24 and 48 h, according to previously described procedures (Stevens & Kaplan, 2000; Martin et al., 2002). Any ß-haemolytic colonies were subcultured, isolated and typed (PathoDx).

Acute S. pyogenes infection was diagnosed by the presence of S. pyogenes in throat cultures of patients with acute pharyngitis; presence of S. pyogenes in throat cultures of healthy controls was considered to be a carrier state.

Statistical analysis.

Data entry and statistical analyses were carried out by using SAS software (version 8.0; SAS Institute). A P value of <0.05 was considered to be statistically significant for all statistical tests. Parametric data were compared by using analysis of variance (ANOVA) with terms for treatment and tests for multiple comparisons; when data were not normally distributed or were non-parametric, the Kruskal–Wallis test was used. Categorical data were analysed by using contingency analysis and a S² or Fisher's test.

	RESULTS

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Bacterial and viral findings

At least one of the bacteria or viruses studied was found in 103 patients (81.1 %) and 31 controls (23.8 %; P < 0.0001) (Table 1). Viruses were demonstrated in 43 patients (33.8 %) and five controls (3.8 %; P < 0.0001), bacteria in 34 patients (26.8 %) and 26 controls (20 %; P = 0.256) and mixed viral/bacterial pathogens in 26 patients (20.5 %) and none of the controls (P < 0.0001). The agents found most frequently were adenovirus (34 patients versus four controls; P < 0.0001), RSV (27 patients versus one control; P < 0.0001), M. pneumoniae (25 patients versus three controls; P < 0.0001), S. pyogenes (24 patients versus 21 controls; P = 0.678) and C. pneumoniae (17 patients versus two controls; P = 0.0006). Acute M. pneumoniae infection was determined serologically in all infected subjects (specific IgM 1 : 100 in 21 patients and two controls, and a fourfold increase in IgG in four patients and one control), and confirmed by PCR in 16 patients (64 %) and none of the controls. Acute C. pneumoniae infection was determined serologically in 10 of 17 infected patients and in the two infected controls (all with a fourfold increase in IgG; none was positive for IgM), and confirmed by PCR in six patients (60 %) and none of the controls. C. pneumoniae DNA was detected in a further seven patients, who showed no serological signs of acute infection.

A single pathogen was demonstrated in 65 patients (51.1 %) and 31 controls (23.8 %; P < 0.0001), whereas two pathogens were diagnosed in 38 patients (29.9 %) and none of the controls (P < 0.0001). Among patients, M. pneumoniae was the single pathogen that was found most frequently (18/25 cases of M. pneumoniae infection, 72 %), followed by adenovirus (16/34 cases of adenoviral infection, 47.1 %) and RSV (10/27 cases of RSV infection, 37 %). S. pyogenes and C. pneumoniae were found as single pathogens in a minority of patients [6/24 cases of S. pyogenes infection (25 %) and 4/17 cases of C. pneumoniae infection (23.5 %)].

Clinical and laboratory findings

Table 2 shows the epidemiological and clinical characteristics of patients with acute pharyngitis and healthy controls in whom a single infectious agent was demonstrated. A history of recurrent pharyngitis and having older siblings were significantly more frequent in patients with acute M. pneumoniae infection than in those with pharyngitis associated with other infections and healthy controls. No other significant difference between groups of patients was observed; none of the controls had signs or symptoms of acute pharyngitis. Similar results were obtained when patients with infections due to M. pneumoniae together with other pathogens were compared with other multiple infections, thus confirming the relationship between M. pneumoniae and history of recurrent pharyngitis and having older siblings.

Table 3 summarizes the laboratory data at enrolment in patients with acute pharyngitis and healthy controls in whom a single infectious agent was demonstrated. There were no significant differences between patients with pharyngitis due to the various infectious agents and these parameters were also not significantly different among patients with multiple infections. Total white blood cell counts, C-reactive protein concentrations and erythrocyte sedimentation rates were significantly higher in all groups of patients with pharyngitis than in healthy controls.

Clinical outcome

Table 4 compares the clinical outcome of patients with acute pharyngitis and healthy controls in whom a single infectious agent was demonstrated. Duration of fever was significantly longer in patients with acute M. pneumoniae infection than in those with infections due to other pathogens. There were no significant inter-group differences in duration of the other symptoms of pharyngitis, but there was a trend toward longer persistence of all symptoms among patients with M. pneumoniae infection. A negative outcome, i.e. recurrence of acute pharyngitis after an initial cure, was significantly more frequent among patients with acute M. pneumoniae infection than among those with infections due to other single pathogens. No patient developed different respiratory tract infections or progressed to more serious lower respiratory tract disease or other systemic infection during the study period. Similar results were observed when patients with infections due to M. pneumoniae plus other pathogens were compared with those with other multiple infections. None of the healthy controls showed any signs or symptoms of disease during the study period.

	DISCUSSION

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
This study highlights the prominent role of viruses, mainly adenovirus and RSV, in acute childhood pharyngitis (Putto et al., 1986; Putto, 1987; Tsai et al., 2001; Nokso-Koivisto et al., 2002). It also shows that, among the bacteria studied, S. pyogenes is found frequently in acute pharyngitis (Bisno et al., 1997, 2002; Bisno, 2001) but, as it is often present together with other viruses or bacteria that are involved in the aetiology of the disease and may also be present in healthy subjects, it is not possible to distinguish carriers from patients with a true infection. Finally, our results indicate that atypical bacteria play a role in causing acute pharyngitis, but show that this is much more relevant for M. pneumoniae than for C. pneumoniae. These data, supported by the findings that atypical bacterial infections are very rare in healthy controls, suggest that M. pneumoniae is able to cause acute pharyngitis per se, whereas C. pneumoniae seems to be mainly a co-pathogen.

The importance of M. pneumoniae as a primary cause of acute pharyngitis appears to be further demonstrated by the history and clinical outcome in patients with this infection. Previous recurrent episodes of pharyngitis seem to be specific for acute M. pneumoniae infection; this may be considered to be a useful parameter for differentiating M. pneumoniae from other pathogens in acute pharyngitis. Similar data have been observed in wheezing; a history of recurrent wheezing is significantly more frequent in patients infected by M. pneumoniae (Esposito et al., 2000). Presence of older siblings also seems to be more common in children with acute M. pneumoniae infection, a finding that concurs with data that show that transmission of the pathogen occurs frequently in the household, with school-age children being the main reservoir (Dorigo-Zetsma et al., 2001). Moreover, most episodes of non-streptococcal pharyngitis associated with infections due to C. pneumoniae and/or viruses resolved spontaneously in our patients and did not relapse, despite absence of antibiotic treatment, whereas a significant proportion of pharyngitis associated with M. pneumoniae infections had a negative course, with longer duration of fever and recurrence of symptoms within a short time. This seems to confirm the role of M. pneumoniae in the pathogenesis of pharyngitis and suggests that antibiotic therapy is required for M. pneumoniae infection, in order to minimize the risk of recurrence and reduce transmission of the pathogen within households.

Unfortunately, none of the clinical or laboratory findings in children with acute pharyngitis was associated with a particular aetiological agent and none was sufficient to distinguish one infection from another. However, it is well-known that the presence of S. pyogenes cannot be diagnosed on the basis of presenting manifestations or non-specific laboratory findings, but can only be identified by means of a throat culture or rapid antigen detection test (Putto et al., 1986; Putto, 1987; Bisno et al., 1997, 2002; Bisno, 2001). However, diagnosis of acute M. pneumoniae infection currently depends on non-routine laboratory methods (File et al., 1998; Layani-Milon et al., 1999; Apfalter et al., 2001; Dowell et al., 2001; Esposito & Principi, 2002), which make it difficult to identify in outpatient practice. To avoid risk of an inappropriate therapeutic approach to acute pharyngitis, simple laboratory investigations that allow rapid identification of infections due to M. pneumoniae are urgently needed.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
We thank Maria Teresa Panza for her contribution to this study. This work was supported in part by an educational grant from Abbott Spa, Italy.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES