Being directly exposed to the external environment, the nasopharynx of children has resident microbial flora that usually does not harm the subject but which, in some cases, constitutes the reservoir of pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis) implicated in upper respiratory tract infections, including otitis media and sinusitis.1-10
Bacteria carried in the nasopharynx of normal children reflect the strains currently circulating in the community and causing respiratory infections; therefore study of the prevalence of these bacteria and of their resistance patterns can provide useful indications for more rational antibiotic therapy of respiratory infections.11, 12 Such evaluation is even more important if one considers that in recent years the emergence of resistance to beta-lactam drugs among respiratory pathogens has become a major worldwide therapeutic problem13-15 and that, at least in Italy, a substantial increase has been demonstrated in the local rates of erythromycin resistance in S. pneumoniae strains obtained from adults and children with various diseases.13, 16
We report the results of the Ascanius Project which was established in Italy in 1996 with the following aims: (1) to assess the prevalence of respiratory pathogens (S. pneumoniae, H. influenzae and M. catarrhalis) in the nasopharynx of healthy children attending day-care centers and primary schools; (2) to evaluate the risk factors for the carrier status; and (3) to determine the antimicrobial susceptibility of a wide range of therapeutic compounds, commonly prescribed for pediatric respiratory infections.
PATIENTS AND METHODS
Study population. From October 15, 1996, to November 15, 1996, a single nasopharyngeal specimen was obtained from children attending day-care centers and the first years of primary school in 18 cities in Italy (10 in Northern, 3 in Central and 5 in Southern Italy, respectively), with populations ranging from 20 000 to 500 000 inhabitants. In each city only one pediatrician, belonging to the main Pediatric Department, was responsible for the study whereas 2 day-care centers (1 for infants and children up to 3 years of age, 1 for children ages 4 to 5 years) and 1 primary school (children 6 to 7 years) were included. All children attending each participating center were eligible for enrollment unless they had an underlying chronic illness (immunologic diseases; neoplastic diseases; renal, cardiac, hepatic or hematologic diseases; bronchodysplasia; Down's syndrome; chronic otitis media with effusion) or an acute upper or lower respiratory tract infection (rhinitis, nasopharyngitis, tonsillitis, acute otitis media, laryngitis, tracheobronchitis), even if mild.
The research protocol was approved by the Ethics Committees of the Pediatric Departments in charge of health control of the day-care centers and schools in each city. Signed informed consent was obtained from a parent or guardian of each child. Written questionnaires for obtaining information about demographics and medical history were completed by the parent(s) in the presence of a pediatrician. Questions included: (1) duration of breast-feeding: (2) duration of previous day-care attendance; (3) the number of family members and their age; (4) birth rank; (5) smoking habits of all the family members living together; (6) living conditions (urban area, i.e. town, or rural area, i.e. country) and, regarding the previous 3 months (7) the number and type of respiratory infections (including otitis media, tonsillitis, sinusitis, rhinitis, pneumonia); (8) the number and type of antimicrobial drugs administered; and (9) the number and reason of hospitalizations. When possible, in addition to the parenterally recalled information, the interviewing pediatrician collected data on each child's medical history in the child's health booklet kept in the day-care center or school and in the hospital discharge record. All information was gathered without knowledge of the child's carrier status.
Specimen collection. Nasopharyngeal specimens were obtained by a single trained investigator in each center, following a protocol established before the study. In particular a Mini-Culturette (Becton Dickinson, Cockeysville, MD) extra thin flexible wire swab, with the tip bent at approximately a 30-degree angle, inserted through the mouth and placed 1 to 1.5 inches into the nasopharynx without touching the uvula or the tongue and maintained in place for at least 5 s. Children were not allowed to eat or drink within 3 h before specimen collection.
Microbiology procedures. The same microbiology procedures were utilized by all the participating centers, following guidelines validated by the chief microbiologist (GCS). Nasopharyngeal cultures were inoculated into Stuart (Venturi Transystem, Brescia, Italy) transport medium tubes, which were transported to the Microbiology Laboratory, located in the same hospital of each Pediatric Department involved, within 1 to 3 h from culture collection and processed immediately. S. pneumoniae, H. influenzae and M. catarrhalis were isolated and identified by standard laboratory procedures. In each site strains were stored in serumglycerol freezing medium, frozen at −80°C; afterward the frozen strains were transported to the Institute of Microbiology, University of Genoa for confirmation of identity and antimicrobial susceptibility testing. In particular the identity was confirmed by the following procedures: (1) H. influenzae: Gram stain morphology, X (hemin), V (nicotinamide adenine dinucleotide) and combined XV factor dependency; (2) S. pneumoniae: Gram stain morphology, catalase reaction, optochin susceptibility and bile solubility; (3) M. catarrhalis: Gram stain morphology, catalase reaction and hydrolysis of tributyrin. The MICs of the antimicrobials were determined by the Sensititre microbroth incorporation technique with an inoculum of approximately 105 colony-forming units/ml. The medium used was Mueller-Hinton broth, supplemented with saponin-lysed horse blood and Factor V for isolates of Haemophilus spp. and S. pneumoniae. The choice of antimicrobial agents and the concentration tested were as follows: penicillin (0.03 to 16 mg/l); amoxicillin (0.06 to 32 mg/l); amoxicillin-clavulanic acid (0.06 to 32 mg/l); cefaclor (0.25 to 64 mg/l); cefuroxime (0.25 to 64 mg/l); cefixime (0.12 to 32 mg/l); ceftriaxone (0.12 to 64 mg/l); erythromycin (0.06 to 32 mg/l); clarithromycin (0.06 to 32 mg/l); azithromycin (0.06 to 32 mg/l); chloramphenicol (0.25 to 64 mg/l). In the case of S. pneumoniae, retesting with beta-lactams was performed to determine the precise MIC endpoints and ampicillin was added to the panel of agents. The range of concentrations tested was as follows: penicillin (0.004 to 8 mg/l); ampicillin (0.004 to 8 mg/l); amoxicillin (0.004 to 8 mg/l); amoxicillin-clavulanic acid (0.004 to 8 mg/l); cefaclor (0.03 to 64 mg/l); cefixime (0.03 to 64 mg/l); cefuroxime (0.015 to 32 mg/l); and ceftriaxone (0.004 to 4 mg/l). Further details on the microbiologic methodology used are reported in the article describing the results of the Alexander Project.17
Data management and analysis. Information was registered in precoded forms and analyzed with the SAS release 6.12 program (SAS Institute, Cary, NC). Quantitative variables were compared by Student's test and, if necessary, qualitative variables were compared by the chi square test with Yates correction. Odds ratio and relative confidence intervals were provided for variables possibly related to nasopharyngeal carriage. The adjusted odds ratios were estimated in logistic regression modeling based on all potential risk factors identified in stratified analysis, eliminating one risk factor at a time with a significance level above 0.15 until only significant risk factors were left in the model. All reported P values are two sided and referred to the significance level of 5%.
Study participation. A total of 1723 children were enrolled in the study. Table 1 summarizes the demographic and clinical characteristics of the study population. The median number of patients enrolled in each center was 99 (range, 73 to 113). Most children were boys, belonged to small families, attended a large day-care center full-time and had had none or only one upper respiratory tract infection in the previous 3 months. About 20% of the children lived in a rural area but attended day care or school in the small town nearby. No child had received H. influenzae type b vaccine.
Recovery of respiratory pathogens from nasopharyngeal cultures. The overall carrier rate of respiratory pathogens was 17.9% (309 of 1723) (Table 2). Most of the children (281, 90.9%) carried only one respiratory pathogen. No child carried 3 respiratory pathogens. In 78 children (25.2%) respiratory pathogens were carried together with Streptococcus pyogenes (23, 7.4%), Staphylococcus aureus (45, 14.6%) or both (10, 3.2%).
The proportion of children carrying respiratory pathogens was significantly higher in children 1 to 3 years old than in those 6 years of age or older. The most frequently carried single respiratory pathogen was H. influenzae in all the age groups. The prevalence of H. influenzae and M. catarrhalis decreased significantly as age increased.
Among the 1414 children who did not carry respiratory pathogens, 265 (18.7%) carried S. aureus, 76 (5.4%) carried S. pyogenes and 21 (1.5%) carried S. aureus and S. pyogenes together.
Susceptibility patterns. Only 3 (5%) isolates of S. pneumoniae were penicillin-resistant (2 intermediately resistant, MIC 0.1 to 1.0 mg/l; and 1 fully resistant, MIC ≥ 2 mg/l). On the contrary resistance to erythromycin, clarithromycin and azithromycin (MIC50 0.25, MIC90 ≥ 64) was very common (41.6%, 25 isolates) among S. pneumoniae isolates. Resistance to ceftriaxone was not detected. Only one isolate was resistant both to penicillin and to macrolides. Beta-lactamase production was present in ∼6% (12) of H. influenzae isolates and in 88.7% (63) of M. catarrhalis isolates.
Investigation of risk factors for nasopharyngeal carriage of respiratory pathogens. Univariate analysis of factors potentially associated with nasopharyngeal carriage of respiratory pathogens indicated as significant having older sibling(s) (odds ratio, 1.31; P = 0.04), living in a rural area (odds ratio, 0.71; P = 0.04) and previous day-care attendance (full-time for up to or more than 1 year, in a large day-care center) (odds ratio, 1.18; P = 0.02). Variables nonsignificantly associated with nasopharyngeal carriage, either considering all pathogens together or single species or different age groups, were: sex; past breast-feeding (≥3 months); exposure to passive smoking (both considering less or more than 10 cigarettes/day); having been hospitalized; or having had one or more upper respiratory infections in the previous 3 months.
Multivariate analysis of the variables potentially associated with risk of nasopharyngeal carriage of respiratory pathogens, considered overall, indicated as significant age ≤3 years, having one or more older siblings and history of prolonged day-care attendance; whereas living in a rural area had a protective effect against carriage (Table 3). After distinguishing among the different pathogens, previous day-care attendance remained significant for all pathogens, young age was significantly associated with carriage of M. catarrhalis and living in a rural area was protective against carriage of S. pneumoniae and H. influenzae.
Considering all respiratory pathogens together but stratifying children according to age groups, in children 1 to 3 years of age previous day-care attendance (odds ratio, 1.29; P = 0.006) and having an older sibling (odds ratio, 1.29; P = 0.03) were still significant variables. In children ages 4 to 5 years variables associated with nasopharyngeal carriage were previous day-care attendance (odds ratio, 1.30; P = 0.04) and living in a rural area (odds ratio, 0.357; P = 0.03). In children 6 to 7 years old no significant association was found.
Antibiotic therapy as risk factor for carriage of respiratory pathogens. About one-fourth of the enrolled children had received one or more courses of antibiotic therapy in the previous 3 months. No child had received antibiotic prophylaxis. Cephalosporins were the most prescribed drugs and were given orally in >98% of the cases. The most frequent cephalosporin was cefaclor, followed by cefixime and ceftibuten. First generation cephalosporins were not used. Macrolides were the second most frequent class of drugs administered; clarithromycin and azithromycin were given in ∼75% of the cases. Aminopenicillins included mainly amoxicillin (84.5% of the courses).
As regards nasopharyngeal carriage of respiratory pathogens overall, the proportion of children given one or more courses of antibiotic therapy was not different in carriers compared with noncarriers (Table 4). Moreover no significant differences were found between carriers and noncarriers as regards the type of drugs used.
When considering single pathogens, the proportion of children given any previous antibiotic therapy/ies was not different among carriers compared with non-carriers. The use of aminopenicillins, amoxicillin plus clavulanic acid or cephalosporins was not associated with a greater risk of nasopharyngeal carriage of any respiratory pathogen, either considering the type of drug or the number of courses. On the contrary repeated treatments with a macrolide were significantly associated with nasopharyngeal carriage of S. pneumoniae (P = 0.03; multivariate analysis: odds ratio, 1.74; P = 0.03). Among these isolates erythromycin resistance was present in four of seven strains (57.1%).
The prevalence of nasopharyngeal carriage of respiratory pathogens in Italian children is quite low: only about one-fifth of the children harbored one respiratory pathogen and very few carried two pathogens in the nasopharynx. Even if similar low carriage rates had been reported for single respiratory pathogens,9, 18 our findings differ from most of the studies on nasopharyngeal carriage, which reported colonization rates ranging from 20 to >50% for all respiratory pathogens.6, 7, 10, 19, 20 In our opinion there are several explanations for our observation. (1) In contrast to many of the studies7, 21, 22 our survey was made only in healthy children. Because during episodes of respiratory illness, in particular otitis media, nasopharyngeal flora remarkably increases, the inclusion of ill children with healthy ones, often found in the literature, may result in a higher carriage rate.7, 23, 24 (2) Season may be important. Children were sampled during a very short period of time, in midautumn, in contrast to most studies, in which sampling was performed throughout several months, involving different seasons.10, 18, 22, 25 Our study design might have avoided the possible effect of previous upper respiratory tract infections, which were infrequent in the immediately previous months, July to September. Moreover only a very minor proportion of children had been previously hospitalized. (3) We studied a very large number of children, which is likely representative of these age groups and geographic site. In contrast most previous studies included only selected groups of children or small samples, with the risk of focusing on specific situations, not really generalizable. (4) Human genetic traits might play a role. Sung et al.9 showed remarkably lower nasopharygeal carriage in Chinese children compared with Vietnamese children living the same locality and in the same crowding conditions. In addition a sampling or laboratory error is not likely, because there was an accurate pretraining of all the investigators and microbiologic procedures were monitored throughout the study.
Regarding susceptibility patterns, in our survey the rate of penicillin resistance among S. pneumoniae isolates and beta-lactamase production among H. influenzae strains was very low. These findings are in contrast to data reported in other European countries but are consistent with previous Italian reports regarding adults and children with lower respiratory tract infections.13, 14 The data regarding erythromycin resistance of S. pneumoniae are in agreement with previous Italian data that showed an increasing macrolide resistance among strains of S. pneumoniae collected from hospitalized subjects, reaching 23% in 1996.15, 16
To our knowledge our study is the largest one evaluating a great number of risk factors for nasopharyngeal carriage of all three potential respiratory pathogens. Children younger than 3 years were more frequently carriers than older children. Our data are generally in agreement with those of Ingvarsson et al.,6 who showed that the rate of carriage of S. pneumoniae and M. catarrhalis is high in infancy and tends to decrease with age. The lower frequency of H. influenzae in children 6 to 7 years old is in contrast with Ingvarsson's data: children in our survey might have developed natural immunity and a consequent reduced colonization26 or, given that H. influenzae exhibits the highest carriage rate during illness, the relative infrequency of previous upper respiratory infections in this age group might have accounted for the low carriage rate.3
As regards exposure to other children, our data are consistent with the literature; the role of day-care attendance as risk factor is remarkable only when the attendance is full-time, continuous and prolonged and its importance is greater in younger children than in older ones.10, 19, 27-31 The long term impact of previous day-care attendance on nasopharyngeal carriage has not been described before; it might reflect the long term effect of recurrent respiratory infections typically occurring in day-care centers and is consistent with the data of Faden et al.,5 who found that children with a history of recurrent otitis media, even when healthy, carried respiratory pathogens more frequently than normal children. Moreover the relationship between the extent of exposure to other children and nasopharyngeal carriage of respiratory pathogens is further confirmed by the protective effect of living in a rural, less crowded area and by the association with the presence of older siblings, especially in the youngest age group, already described in a few studies.10, 19, 32
A recent (≤3 months) diagnosis of one or more upper respiratory tract infections was not associated with nasopharyngeal carriage of respiratory pathogens. However, the high proportion of children who had none or only one previous episode probably precludes a definitive evaluation of this variable. In addition past breast-feeding and exposure to passive smoking were not associated with the presence of respiratory pathogens. The data regarding breast-feeding are still controversial7, 33, 34 and further studies are needed. To our knowledge no studies have thus far explored the role of passive smoking in nasopharyngeal carriage.
We did not find any relationship between antibiotic use per se or the type of drug used in the previous 3 months and nasopharyngeal carriage of pathogens, considering all bacteria together. In contrast, considering each pathogen alone, repeated courses of macrolides in the previous 3 months were significantly associated with carriage of S. pneumoniae strains characterized by a higher prevalence of resistance to erythromycin than that of the overall isolated S. pneumoniae. Despite the limitation related to the small sample, our data corroborate findings of other studies that have demonstrated an association between the use of a specific antibiotic and selective colonization with strains resistant to that drug.18, 22, 27, 35, 36 Most of the studies actually available have warned about the risk of carriage of penicillin-resistant S. pneumoniae in children previously treated with drugs to which the pathogen was highly resistant. However, in a different microbiologic scenery, in which macrolide resistance is the actual problem, one should principally be cautious in using this group of drugs. Moreover one should be aware that the number of courses of drugs to which pathogens are resistant has the utmost importance. What appears to matter most is not whether a drug is used but how many times in the same subject.
In conclusion our study shows that the number of children to whom a child is exposed in the first years of life, whether in day care or at home, and the duration of exposure has a strong and long term relationship with nasopharyngeal carriage of respiratory pathogens. Our data might encourage changes in day-care organizations, favoring small day-care centers and/or part time attendance. In addition, because repeated exposure to antimicrobials to which respiratory pathogens are resistant is strongly associated with nasopharyngeal carriage, antimicrobial restrictive strategies should be tailored to local microbiologic sceneries.
This work was supported in part by an educational grant by SmithKline Beecham Pharmaceuticals, Italy.
The Ascanius Project Collaborative Group includes E. Reali (Cinisello Balsamo, Italy), R. Longhi (Como, Italy), G. L. Marseglia (Pavia, Italy), D. Pavesio (Turin, Italy), G. Bona (Novara, Italy), B. Sacher (S. Daniele Friuli, Italy), M. De Santis (Genoa, Italy), G. P. Salvioli (Bologne, Italy), G. Dodi (Fidenza, Italy), G. Farneti (Cesena, Italy), G. Caramia (Ancona, Italy), P. Baroncelli (Pisa, Italy), T. Meloni (Sassari, Italy), A. Guarino (Naples, Italy), A. Russo (Bari, Italy), F. Pusceddu (Cagliari, Italy), L. Ziino (Palermo, Italy) and G. Salpietro (Messina, Italy).
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